Spatially-tagged analyte capture agents for analyte multiplexing

ABSTRACT

Provided herein are methods for preparing biological samples for spatial proteomic analysis, methods of determining a location of a protein analyte in a biological sample, and methods of determining a location of a protein analyte and a nucleic acid analyte in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to: International Application No.PCT/US2020/049048, filed Sep. 2, 2020, which claims the benefit of U.S.Provisional Patent Application No. 62/933,299, filed Nov. 8, 2019, U.S.Provisional Patent Application No. 62/939,488, filed Nov. 22, 2019, U.S.Provisional Patent Application No. 62/959,526, filed Jan. 10, 2020, U.S.Provisional Patent Application No. 63/000,313, filed Mar. 26, 2020, U.S.Provisional Patent Application No. 63/019,209, filed May 1, 2020, andU.S. Provisional Patent Application No. 63/039,928, filed Jun. 16, 2020.The contents of each of these applications are incorporated by referencein their entireties.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the context of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

The spatial organization of gene expression can be observed within asingle cell, tissue, or organism. Genetic material and related gene andprotein expression influences cellular fate and behavior. Although thespatial heterogeneity in developing systems has been studied, someapproaches, however, currently rely on a small set of pre-definedmarkers, therefore introducing selection bias that limits discovery andmaking it costly and laborious to localize RNA transcriptome-wide.

SUMMARY

Provided herein are methods for preparing a biological sample forspatial proteomic analysis comprising (a) placing the biological sampleon a substrate, wherein the substrate comprises a plurality of captureprobes, wherein a capture probe of the plurality of capture probescomprises a spatial barcode and a capture domain; (b) contacting thebiological sample with a protein blocking buffer; and (c) contacting thebiological sample with a plurality of analyte capture agents, wherein ananalyte capture agent of the plurality of analyte capture agentscomprises: (i) an analyte binding moiety that binds specifically to ananalyte from the biological sample; (ii) an analyte binding moietybarcode; and (iii) an analyte capture sequence, wherein the analytecapture sequence binds specifically to the capture domain of the captureprobe.

In some embodiments, the analyte from the biological sample is aprotein.

In some embodiments, the method further comprises (d) contacting thebiological sample with methanol for about 5 minutes to about 30 minutesat about −30° C. to about 0° C.

In some embodiments, step (d) is performed before step (a); step (a) isperformed before step (d); or step (d) is performed between step (a) andstep (b).

In some embodiments the method further comprises, after step (b): (e)contacting the biological sample with a fluorescently-labeled antibody.

In some embodiments, step (e) is performed between steps (b) and (c);step (e) is performed simultaneously with step (c); or step (e) isperformed after step (c).

In some embodiments, the method further comprises, after step (e): (f)performing fluorescence imaging of the biological sample.

In some embodiments, step (f) is performed before step (c); or step (f)is performed after step (c).

In some embodiments, the method further comprises, after step (c): (g)permeabilizing the biological sample.

In some embodiments, step (g) is performed after step (f).

In some embodiments, the method further comprises, before step (f): (h)contacting the biological sample with a coverslip.

In some embodiments, the analyte binding moiety is an antibody orantigen-binding fragment thereof, a cell surface receptor bindingmolecule, a receptor ligand, a small molecule, a T-cell receptorengager, a B-cell receptor engager, a pro-body, an aptamer, a monobody,an affimer, or a darpin.

In some embodiments, the protein blocking buffer comprises a first RNAseinhibitor, a second RNAse inhibitor, an acid salt buffer, a non-specificblocking protein, and a surfactant.

In some embodiments, the first RNAse inhibitor comprises ribonucleotidevandyl complex. In some embodiments, the ribonucleoside vandyl complexis present in the protein blocking buffer in an amount of about 5 mM toabout 50 mM.

In some embodiments, the non-specific blocking protein is present in theprotein blocking buffer in an amount of about 1% to about 3% (w/v). Insome embodiments, the non-specific blocking protein is a serum albumin.In some embodiments, the serum albumin is bovine serum albumin.

In some embodiments, the surfactant is a non-ionic surfactant. In someembodiments, the surfactant is (C₁₄H₂₂O(C₂H₄O)_(n)). In someembodiments, the surfactant is present in the protein blocking buffer inan amount of about 0.05% to about 0.2% (w/v).

In some embodiments, the second RNAse inhibitor is present in theprotein blocking buffer in an amount of about 0.5 U/μL to about 5 U/μL.

In some embodiments, the acid salt buffer is saline sodium citrate.

In some embodiments, the biological sample is contacted with the proteinblocking buffer for about 1 minute to about 10 minutes. In someembodiments, the biological sample is contacted with the proteinblocking buffer at a temperature of about 1° C. to about 25° C.

In some embodiments, the capture probe further comprises a uniquemolecular identifier. In some embodiments, the capture domain comprisesa poly(T) sequence. In some embodiments, the analyte capture sequencecomprises a poly(A) sequence.

In some embodiments, the biological sample is a tissue sample. In someembodiments, the tissue sample is a tissue section.

In some embodiments, the substrate comprises a slide or a bead.

Also provided herein are methods of determining a location of a proteinanalyte in a biological sample comprising preparing a biological sampleaccording to any one of the methods described herein; determining (i) asequence corresponding to the analyte binding moiety barcode of theanalyte binding agent or a complement thereof, and (ii) a sequencecorresponding to the spatial barcode of the capture probe or acomplement thereof, and using the determined sequences of (i) and (ii)to determine the location of the protein analyte in the biologicalsample.

In some embodiments, the determining of the sequences of (i) and (ii)comprises sequencing the sequences of (i) and (ii).

Also provided herein are methods of determining a location of a proteinanalyte and a nucleic acid analyte in a biological sample comprising (1)preparing a biological sample according to any one of the methodsdisclosed herein, wherein preparing the biological sample comprisespermeabilizing the biological sample, the analyte from the biologicalsample that is specifically bound by the analyte binding moiety of theanalyte capture agent is the protein analyte, and the substratecomprises a second capture probe of the plurality of capture probes,wherein the second capture probe comprises a spatial barcode and acapture domain, wherein the capture domain of the second capture probebinds specifically to the nucleic acid analyte; (2) determining (i) asequence corresponding to the analyte binding domain barcode or acomplement thereof, and (ii) a sequence corresponding to the spatialbarcode of the capture probe or a complement thereof, and using thedetermined sequences of (i) and (ii) to determine the location of theprotein analyte in the biological sample; and (3) determining (i) asequence corresponding to the nucleic acid analyte or a complementthereof, and (ii) a sequence corresponding to the spatial barcode of thesecond capture probe or a complement thereof, and using the determinedsequences of (i) and (ii) to determine the location of the nucleic acidanalyte in the biological sample.

In some embodiments, step (2) is performed before step (3); step (2) isperformed after step (3); or step (2) is performed simultaneously withstep (3).

In some embodiments, the method further comprises contacting thebiological sample with a fluorescently-labeled antibody.

In some embodiments, the analyte binding moiety is an antibody orantigen-binding fragment thereof, a cell surface receptor bindingmolecule, a receptor ligand, a small molecule, a T-cell receptorengager, a B-cell receptor engager, a pro-body, an aptamer, a monobody,an affimer, or a darpin.

All publications, patents, patent applications, and informationavailable on the internet and mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication, patent, patent application, or item of information wasspecifically and individually indicated to be incorporated by reference.To the extent publications, patents, patent applications, and items ofinformation incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 shows an exemplary spatial analysis workflow.

FIG. 2 shows an exemplary spatial analysis workflow.

FIG. 3 shows an exemplary spatial analysis workflow.

FIG. 4 shows an exemplary spatial analysis workflow.

FIG. 5 shows an exemplary spatial analysis workflow.

FIG. 6 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 7 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 8 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature.

FIG. 9 is a schematic diagram of an exemplary analyte capture agent.

FIG. 10 is a schematic diagram depicting an exemplary interactionbetween a feature-immobilized capture probe 1024 and an analyte captureagent 1026.

FIGS. 11A, 11B, and 11C are schematics illustrating how streptavidincell tags can be utilized in an array-based system to produce aspatially-barcoded cells or cellular contents.

FIG. 12 is a schematic showing the arrangement of barcoded featureswithin an array.

FIG. 13 is a schematic illustrating a side view of a diffusion-resistantmedium, e.g., a lid.

FIGS. 14A and 14B are schematics illustrating expanded FIG. 14A and sideviews FIG. 14B of an electrophoretic transfer system configured todirect transcript analytes toward a spatially-barcoded capture probearray.

FIG. 15 is a schematic illustrating an exemplary workflow protocolutilizing an electrophoretic transfer system.

FIG. 16 shows an example of a microfluidic channel structure 1600 forpartitioning dissociated sample (e.g., biological particles orindividual cells from a sample).

FIG. 17A shows an example of a microfluidic channel structure 1700 fordelivering spatial barcode carrying beads to droplets.

FIG. 17B shows a cross-section view of another example of a microfluidicchannel structure 1750 with a geometric feature for controlledpartitioning.

FIG. 17C shows an example of a workflow schematic.

FIG. 18 is a schematic depicting cell tagging using either covalentconjugation of the analyte binding moiety to the cell surface ornon-covalent interactions with cell membrane elements.

FIG. 19 is a schematic depicting cell tagging using eithercell-penetrating peptides or delivery systems.

FIG. 20A is a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for “pixelating” a sample, wherein the sample iscut, stamped, microdissected, or transferred by hollow-needle ormicroneedle, moving a small portion of the sample into an individualpartition or well.

FIG. 20B is a schematic depicting multi-needle pixilation, wherein anarray of needles punched through a sample on a scaffold and intonanowells containing gel beads and reagents below. Once the needle is inthe nanowell, the cell(s) are ejected.

FIG. 21 shows a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for dissociating a spatially-barcoded sample foranalysis via droplet or flow cell analysis methods.

FIG. 22A is a schematic diagram showing an example sample handlingapparatus that can be used to implement various steps and methodsdescribed herein.

FIG. 22B is a schematic diagram showing an example imaging apparatusthat can be used to obtain images of biological samples, analytes, andarrays of features.

FIG. 22C is a schematic diagram of an example of a control unit of theapparatus of FIGS. 22A and 22B.

FIG. 23A shows a histological section of an invasive ductal carcinomaannotated by a pathologist.

FIG. 23B shows a tissue plot with spots colored by unsupervisedclustering.

FIG. 23C is a tSNE plot of spots colored by unsupervised clustering.

FIG. 23D shows a gene expression heat map of the most variable genesbetween 9 clusters.

FIG. 23E shows the expression levels of genes corresponding to humanepidermal growth factor receptor 2 (Her2), estrogen receptor (ER), andprogesterone receptor (PR) in the tissue section.

FIG. 23F shows the expression levels of genes of top differentiallyexpressed genes from each of the 9 clusters on individual plots.

FIG. 23G shows the expression levels of genes of top differentiallyexpressed genes from each of the 9 clusters on a single plot.

FIG. 23H is a plot of the expression levels of the top differentiallyexpressed genes from each of the 9 clusters in invasive ductal cellcarcinoma (IDC) and normal breast tissue.

FIG. 23I shows the expression of KRT14 in IDC and match normal tissue.

FIG. 23J is a plot of the expression levels of extracellular matrixgenes in IDC and normal tissue.

FIG. 24A shows a schematic of an example analytical workflow in whichelectrophoretic migration of analytes is performed afterpermeabilization.

FIG. 24B shows a schematic of an example analytical workflow in whichelectrophoretic migration of analytes and permeabilization are performedsimultaneously.

FIG. 25A shows an example perpendicular, single slide configuration foruse during electrophoresis.

FIG. 25B shows an example parallel, single slide configuration for useduring electrophoresis

FIG. 25C shows an example multi-slide configuration for use duringelectrophoresis.

FIGS. 26A and 26B is a schematic depicting exemplary steps for adding abiological sample to an array.

FIG. 27 is a schematic showing exemplary biological samples that can beused in the methods described herein.

FIG. 28 is a schematic depicting a cell migration assay combined with anarray such as those described herein.

FIG. 29A is a schematic showing a high density array with cells bound tospots having barcoded capture probes and cell capture molecules (e.g., acapture antibody).

FIG. 29B is a schematic showing a portion of a spot on the array of FIG.29A with barcoded capture probes and a capture antibody.

FIG. 30 is a schematic illustrating exemplary steps involved insubcellular fractionation.

FIG. 31 is a schematic illustrating exemplary steps involved insubcellular fractionation.

FIG. 32A is a schematic showing an exemplary substrate and capture probefor detecting a selectively releasing a biological analyte using, forexample, second strand synthesis.

FIG. 32B is a schematic depicting steps for second strand synthesis.

FIG. 33A is a schematic showing an exemplary substrate and captureprobes for detecting a selectively-released biological analyte using,for example, selective probe cleavage.

FIG. 33B is a schematic depicting steps for using selective probecleavage in methods involving detecting a selectively-releasedbiological analyte.

FIG. 34 is a schematic depicting steps for using multiple substrates inmethods involving detecting a selectively-released biological sample.

FIG. 35 is a schematic depicting steps for temporal analysis of abiological analyte.

FIG. 36 shows an exemplary workflow for temporal and spatial profilingof biological analytes at n time-points.

FIG. 37 shows a schematic representation of spatio-temporal profiling ofbiological analytes.

FIG. 38 is a schematic illustrating fluorescently-labeled antibodiesinteracting with analytes within a biological sample, wherein thebiological sample is contacting a capture probe array.

FIG. 39 is a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for immunofluorescently detecting analytes on acapture probe array, wherein the biological sample is fixed, stainedwith fluorescent antibodies, and imaged to detect the spatial locationof target analytes within the biological sample.

FIG. 40 shows exemplary antibody staining wherein tissue sections ofmouse spleen are fixed in either 2% formaldehyde or methanol. In boththe 2% formaldehyde and methanol fixation conditions, CD3 can be seen inthe pockets of white pulp, and CD29 in the red pulp portion of thespleen.

FIGS. 41A and 41B shows exemplary tissue plot of spatially-resolvedinformation, wherein a mouse spleen tissue section is analyzed on abarcoded feature array. Here, analytes are captured on a barcodedfeature array as described herein and captured analytes are clusteredusing a nonlinear dimensionality reduction technique. The clusters canidentify cellular subpopulations within a tissue sample.

FIG. 42 shows exemplary immunofluorescence staining of 2% formaldehydeof mouse spleen on the left, identifying the presence and location ofCD3, CD29, and DAPI. This immunofluorescence staining is overlaid withCD3 UMI counts (right), obtained by performing the spatial workflowsdescribed herein. The CD3 UMI counts (right) demonstrates that specificanalytes can be captured and barcoded according to the spatial workflowsdescribed herein, wherein the spatial workflows are compatible with andcomplementary to other staining/imaging techniques (e.g.,immunofluorescence).

FIG. 43 shows exemplary immunofluorescence staining of methanol of mousespleen on the left, identifying the presence and location of CD3, CD29,and DAPI. This immunofluorescence staining is overlaid with CD3 UMIcounts (right), obtained by performing the spatial workflows describedherein. The CD3 UMI counts (right) demonstrates that specific analytescan be captured and barcoded according to the spatial workflowsdescribed herein, wherein the spatial workflows are compatible with andcomplementary to other staining/imaging techniques (e.g.,immunofluorescence).

FIG. 44 shows exemplary immunofluorescence staining of 2% formaldehydeand methanol fixation of mouse brain in three channels (NeuN; GFAP;DAPI).

FIGS. 45A and 45B shows exemplary UMI and cluster data of mouse brainfor 2% formaldehyde (FIG. 43A) and methanol (FIG. 43B) fixationconditions, wherein clusters were determined using t-SNE to identifydifferent cell types.

FIG. 46 shows exemplary immunofluorescence staining of 2% formaldehydefixation of mouse brain on the left. Right panels showimmunofluorescence staining overlaid with NeuN UMI counts (top), andGFAP UMI counts (bottom), obtained by performing the spatial workflowsdescribed herein. The NeuN and GFAP UMI counts demonstrates thatspecific analytes can be captured and barcoded according to the spatialworkflows described herein, wherein the spatial workflows are compatiblewith and complementary to other staining/imaging techniques (e.g.,immunofluorescence).

FIG. 47 shows exemplary immunofluorescence staining of methanol fixationof mouse brain on the left. Right panels show immunofluorescencestaining overlaid with NeuN UMI counts (top), and GFAP UMI counts(bottom), obtained by performing the spatial workflows described herein.The NeuN and GFAP UMI counts demonstrates that specific analytes can becaptured and barcoded according to the spatial workflows describedherein, wherein the spatial workflows are compatible with andcomplementary to other staining/imaging techniques (e.g.,immunofluorescence).

FIG. 48 is an exemplary schematic illustrating spatially-tagged analytecapture agents wherein the analyte capture sequence of the capture agentbarcode domain (poly-A sequence) can be blocked with a blocking probe(poly-T oligonucleotude in upper left corner). Once the blocking probeis removed, the spatially-tagged analyte capture agent binds to thecapture probe on the substrate and is labeled with fluorescentantibodies.

FIG. 49 is an exemplary schematic illustrating a spatially-taggedanalyte capture agent wherein the analyte capture sequence is blockedvia a blocking probe. The blocking probe can be removed, e.g., with anRNAse treatment.

FIG. 50 shows exemplary images showing the performance of an oligo (dT)blocking probe under various conditions. The staining was performed witholigo (dT) blocking probes of varying lengths (15-30 nucleotides) at 4°C. and 37° C. and results indicate analyte capture agents were blockedfrom binding to the capture probes at both temperatures, although lowertemperatures appeared more effective when using shorter blocking probes.

FIG. 51 is a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for spatial analyte identification after antibodystaining in a biological sample, wherein the sample is fixed, stainedwith fluorescent antibodies and spatially-tagged analyte capture agents,and imaged to detect the spatial location of target analytes within thebiological sample.

FIG. 52 shows an exemplary image showing immunofluorescence staining forCD29 (left panel) and images showing spatially-resolved informationafter analyzing the capture agent barcode domains for CD29, CD3, CD169,and F4/80 (right panels) in tissue sections of mouse spleen.

FIG. 53 shows exemplary multiplex imaging results, wherein theimmunofluorescent image shows immunofluorescent staining for CD29 andCD4 in tissue sections of mouse spleen (far left). The images in theseries of right panels show the results of a multiplexed,spatially-tagged analyte capture agent workflow, wherein the spatiallocation of target proteins, CD29, CD3, CD4, CD8, CD19, B220, F4/80, andCD169 are visualized by sequencing the analyte-corresponding analytebinding moiety barcodes.

FIG. 54 shows an exemplary workflow for fixing and staining tissuesections using methanol or an aldehyde.

FIG. 55 shows an exemplary workflow for H&E staining of methanol oraldehyde fixed tissues.

FIG. 56 shows an exemplary workflow for immunostaining of methanol oraldehyde fixed tissues.

FIG. 57 shows an exemplary workflow for spatial proteomic and genomicanalysis.

FIG. 58A shows a section of human cerebral cortex stained withhematoxylin and eosin.

FIG. 58B shows a spatial transcriptomic analysis of the section of humancerebral cortex from FIG. 58A, overlaid on FIG. 58A.

FIG. 58C shows a second of human cerebral cortex immunostained forneurofilament H (NFH) and glial fibrillary acidic protein (GFAP).

FIG. 58D shows a spatial transcriptomic analysis of the section of humancerebral cortex from FIG. 58C, overlaid on FIG. 58C.

FIG. 58E shows the spatial distribution of transcription of stathmin-2(STMN2) and RAR related orphan receptor B (RORB) in the sample from FIG.58A, each overlaid on FIG. 58A.

FIG. 58F shows the spatial distribution of transcription of stathmin-2(STMN2) and RAR related orphan receptor B (RORB) in the sample from FIG.58C, each overlaid on FIG. 58C.

FIG. 58G shows immunofluorescence (IF) analysis of glial fibrillaryacidic protein (GFAP) on a section of human cerebral cortex (left) andthe spatial distribution of GFAP overlaid on the IF image (right).

FIG. 58H shows immunofluorescence analysis (IF) of neurofilament H (NFH)on a section of human cerebral cortex (left) and the spatialdistribution of NFEH overlaid on the IF image (right).

FIG. 59A shows a schematic of an analyte capture agent and a spatialgene expression slide.

FIG. 59B shows a merged fluorescent image of DAPI staining of a sectionof human cerebellum tissue.

FIG. 59C shows a spatial transcriptomic analysis of the section of humancerebellum from FIG. 59B, overlaid on FIG. 59B.

FIG. 59D shows a t-SNE projection of the sequencing data illustratingcell-type clustering of the cerebellum from FIG. 59C.

FIG. 59E shows spatial gene expression (top) and protein staining(bottom) of astrocyte marker glutamine synthase (produced by hybridomaclone O91F4), each overlaid on FIG. 59B.

FIG. 59F shows spatial gene expression (top) and protein staining(bottom) of oligodendrocyte marker myelin CNPase (produced by hybridomaclone SMI91), each overlaid on FIG. 59B.

FIG. 59G shows spatial gene expression (top) and protein staining(bottom) of oligodendrocyte marker myelin basic protein (produced byhybridoma clone P82H9), each overlaid on FIG. 59B.

FIG. 59H shows spatial gene expression (top) and protein staining(bottom) of stem cell marker SOX2 (produced by hybridoma clone 14A6A34),each overlaid on FIG. 59B.

FIG. 59I shows spatial gene expression (top) and protein staining(bottom) of neuronal marker SNAP-25 (produced by hybridoma clone SMI81),each overlaid on FIG. 59B.

FIG. 60A shows a merged fluorescent image of DAPI staining of a sectionof a human spinal cord sample.

FIG. 60B shows a spatial transcriptomic analysis of the section ofspinal cord from FIG. 60A, overlaid on FIG. 60A.

FIG. 60C shows spatial gene expression of MBP, overlaid on FIG. 60A.

FIG. 60D shows spatial protein staining of myelin basic protein(produced by hybridoma clone P82H9).

FIG. 60E shows spatial gene expression of SNAP25, overlaid on FIG. 60A.

FIG. 60F shows spatial protein staining of SNAP25.

DETAILED DESCRIPTION I. Introduction

This disclosure describes apparatus, systems, methods, and compositionsfor spatial analysis of biological samples. This section describescertain general terminology, analytes, sample types, and preparativesteps that are referred to in later sections of the disclosure.

(a) Spatial Analysis

Tissues and cells can be obtained from any source. For example, tissuesand cells can be obtained from single-cell or multicellular organisms(e.g., a mammal). Tissues and cells obtained from a mammal, e.g., ahuman, often have varied analyte levels (e.g., gene and/or proteinexpression) which can result in differences in cell morphology and/orfunction. The position of a cell or a subset of cells (e.g., neighboringcells and/or non-neighboring cells) within a tissue can affect, e.g.,the cell's fate, behavior, morphology, and signaling and cross-talk withother cells in the tissue. Information regarding the differences inanalyte levels (gene and/or protein expression) within different cellsin a tissue of a mammal can also help physicians select or administer atreatment that will be effective and can allow researchers to identifyand elucidate differences in cell morphology and/or cell function in thesingle-cell or multicellular organisms (e.g., a mammal) based on thedetected differences in analyte levels within different cells in thetissue. Differences in analyte levels within different cells in a tissueof a mammal can also provide information on how tissues (e.g., healthyand diseased tissues) function and/or develop. Differences in analytelevels within different cells in a tissue of a mammal can also provideinformation of different mechanisms of disease pathogenesis in a tissueand mechanism of action of a therapeutic treatment within a tissue.Differences in analyte levels within different cells in a tissue of amammal can also provide information on drug resistance mechanisms andthe development of the same in a tissue of a mammal. Differences in thepresence or absence of analytes within different cells in a tissue of amulticellular organism (e.g., a mammal) can provide information on drugresistance mechanisms and the development of the same in a tissue of amulticellular organism.

The spatial analysis methodologies herein provide for the detection ofdifferences in an analyte level (e.g., gene and/or protein expression)within different cells in a tissue of a mammal or within a single cellfrom a mammal. For example, spatial analysis methodologies can be usedto detect the differences in analyte levels (e.g., gene and/or proteinexpression) within different cells in histological slide samples, thedata from which can be reassembled to generate a three-dimensional mapof analyte levels (e.g., gene and/or protein expression) of a tissuesample obtained from a mammal, e.g., with a degree of spatial resolution(e.g., single-cell resolution).

Spatial heterogeneity in developing systems has typically been studiedvia RNA hybridization, immunohistochemistry, fluorescent reporters, orpurification or induction of pre-defined subpopulations and subsequentgenomic profiling (e.g., RNA-seq). Such approaches, however, rely on arelatively small set of pre-defined markers, therefore introducingselection bias that limits discovery. These prior approaches also relyon a priori knowledge. RNA assays traditionally relied on staining for alimited number of RNA species. In contrast, single-cell RNA-sequencingallows for deep profiling of cellular gene expression (includingnon-coding RNA), but the established methods separate cells from theirnative spatial context.

Spatial analysis methodologies described herein provide a vast amount ofanalyte level and/or expression data for a variety of multiple analyteswithin a sample at high spatial resolution, e.g., while retaining thenative spatial context. Spatial analysis methods include, e.g., the useof a capture probe including a spatial barcode (e.g., a nucleic acidsequence that provides information as to the position of the captureprobe within a cell or a tissue sample (e.g., mammalian cell or amammalian tissue sample) and a capture domain that is capable of bindingto an analyte (e.g., a protein and/or nucleic acid) produced by and/orpresent in a cell. As described herein, the spatial barcode can be anucleic acid that has a unique sequence, a unique fluorophore or aunique combination of fluorophores, a unique amino acid sequence, aunique heavy metal or a unique combination of heavy metals, or any otherunique detectable agent. The capture domain can be any agent that iscapable of binding to an analyte produced by and/or present in a cell(e.g., a nucleic acid that is capable of hybridizing to a nucleic acidfrom a cell (e.g., an mRNA, genomic DNA, mitochondrial DNA, or miRNA), asubstrate including an analyte, a binding partner of an analyte, or anantibody that binds specifically to an analyte). A capture probe canalso include a nucleic acid sequence that is complementary to a sequenceof a universal forward and/or universal reverse primer. A capture probecan also include a cleavage site (e.g., a cleavage recognition site of arestriction endonuclease), a photolabile bond, a thermosensitive bond,or a chemical-sensitive bond.

The binding of an analyte to a capture probe can be detected using anumber of different methods, e.g., nucleic acid sequencing, fluorophoredetection, nucleic acid amplification, detection of nucleic acidligation, and/or detection of nucleic acid cleavage products. In someexamples, the detection is used to associate a specific spatial barcodewith a specific analyte produced by and/or present in a cell (e.g., amammalian cell).

Capture probes can be, e.g., attached to a surface, e.g., a solid array,a bead, or a coverslip. In some examples, capture probes are notattached to a surface. In some examples, capture probes can beencapsulated within, embedded within, or layered on a surface of apermeable composition (e.g., any of the substrates described herein).For example, capture probes can be encapsulated or disposed within apermeable bead (e.g., a gel bead). In some examples, capture probes canbe encapsulated within, embedded within, or layered on a surface of asubstrate (e.g., any of the exemplary substrates described herein, suchas a hydrogel or a porous membrane).

In some examples, a cell or a tissue sample including a cell arecontacted with capture probes attached to a substrate (e.g., a surfaceof a substrate), and the cell or tissue sample is permeabilized to allowanalytes to be released from the cell and bind to the capture probesattached to the substrate. In some examples, analytes released from acell can be actively directed to the capture probes attached to asubstrate using a variety of methods, e.g., electrophoresis, chemicalgradient, pressure gradient, fluid flow, or magnetic field.

In other examples, a capture probe can be directed to interact with acell or a tissue sample using a variety of methods, e.g., inclusion of alipid anchoring agent in the capture probe, inclusion of an agent thatbinds specifically to, or forms a covalent bond with a membrane proteinin the capture probe, fluid flow, pressure gradient, chemical gradient,or magnetic field.

Non-limiting aspects of spatial analysis methodologies are described inWO2020123305, WO 2011/127099, WO 2014/210233, WO 2014/210225, WO2016/162309, WO 2018/091676, WO 2012/140224, WO 2014/060483, U.S. Pat.Nos. 10,002,316, 9,727,810, U.S. Patent Application Publication No.2017/0016053, Rodrigues et al., Science 363(6434):1463-1467, 2019; WO2018/045186, Lee et al., Nat. Protoc. 10(3):442-458, 2015; WO2016/007839, WO 2018/045181, WO 2014/163886, Trejo et al., PLoS ONE14(2):e0212031, 2019, U.S. Patent Application Publication No.2018/0245142, Chen et al., Science 348(6233):aaa6090, 2015, Gao et al.,BMC Biol. 15:50, 2017, WO 2017/144338, WO 2018/107054, WO 2017/222453,WO 2019/068880, WO 2011/094669, U.S. Pat. Nos. 7,709,198, 8,604,182,8,951,726, 9,783,841, 10,041,949, WO 2016/057552, WO 2017/147483, WO2018/022809, WO 2016/166128, WO 2017/027367, WO 2017/027368, WO2018/136856, WO 2019/075091, U.S. Pat. No. 10,059,990, WO 2018/057999,WO 2015/161173, and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018,and can be used herein in any combination. Further non-limiting aspectsof spatial analysis methodologies are described herein.

(b) General Terminology

Specific terminology is used throughout this disclosure to explainvarious aspects of the apparatus, systems, methods, and compositionsthat are described. This sub-section includes explanations of certainterms that appear in later sections of the disclosure. To the extentthat the descriptions in this section are in apparent conflict withusage in other sections of this disclosure, the definitions in thissection will control.

(i) Barcode

A “barcode” is a label, or identifier, that conveys or is capable ofconveying information (e.g., information about an analyte in a sample, abead, and/or a capture probe). A barcode can be part of an analyte, orindependent of an analyte. A barcode can be attached to an analyte. Aparticular barcode can be unique relative to other barcodes.

Barcodes can have a variety of different formats. For example, barcodescan include non-random, semi-random, and/or random nucleic acid and/oramino acid sequences, and synthetic nucleic acid and/or amino acidsequences. A barcode can be attached to an analyte or to another moietyor structure in a reversible or irreversible manner. A barcode can beadded to, for example, a fragment of a deoxyribonucleic acid (DNA) orribonucleic acid (RNA) sample before or during sequencing of the sample.Barcodes can allow for identification and/or quantification ofindividual sequencing-reads (e.g., a barcode can be or can include aunique molecular identifier or “UMI”).

Barcodes can spatially-resolve molecular components found in biologicalsamples, for example, at single-cell resolution (e.g., a barcode can beor can include a “spatial barcode”). In some embodiments, a barcodeincludes both a UMI and a spatial barcode. In some embodiments, abarcode includes two or more sub-barcodes that together function as asingle barcode (e.g., a polynucleotide barcode). For example, apolynucleotide barcode can include two or more polynucleotide sequences(e.g., sub-barcodes) that may be separated by one or more non-barcodesequences.

(ii) Nucleic Acid and Nucleotide

The terms “nucleic acid” and “nucleotide” are intended to be consistentwith their use in the art and to include naturally-occurring species orfunctional analogs thereof. Particularly useful functional analogs ofnucleic acids are capable of hybridizing to a nucleic acid in asequence-specific fashion (e.g., capable of hybridizing to two nucleicacids such that ligation can occur between the two hybridized nucleicacids) or are capable of being used as a template for replication of aparticular nucleotide sequence. Naturally-occurring nucleic acidsgenerally have a backbone containing phosphodiester bonds. An analogstructure can have an alternate backbone linkage including any of avariety of those known in the art. Naturally-occurring nucleic acidsgenerally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid(DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).

A nucleic acid can contain nucleotides having any of a variety ofanalogs of these sugar moieties that are known in the art. A nucleicacid can include native or non-native nucleotides. In this regard, anative deoxyribonucleic acid can have one or more bases selected fromthe group consisting of adenine (A), thymine (T), cytosine (C), orguanine (G), and a ribonucleic acid can have one or more bases selectedfrom the group consisting of uracil (U), adenine (A), cytosine (C), orguanine (G). Useful non-native bases that can be included in a nucleicacid or nucleotide are known in the art.

(iii) Probe and Target

A “probe” or a “target,” when used in reference to a nucleic acid orsequence of a nucleic acids, is intended as a semantic identifier forthe nucleic acid or sequence in the context of a method or composition,and does not limit the structure or function of the nucleic acid orsequence beyond what is expressly indicated.

(iv) Oligonucleotide and Polynucleotide

The terms “oligonucleotide” and “polynucleotide” are usedinterchangeably to refer to a single-stranded multimer of nucleotidesfrom about 2 to about 500 nucleotides in length. Oligonucleotides can besynthetic, made enzymatically (e.g., via polymerization), or using a“split-pool” method. Oligonucleotides can include ribonucleotidemonomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotidemonomers (i.e., oligodeoxyribonucleotides). In some examples,oligonucleotides can include a combination of both deoxyribonucleotidemonomers and ribonucleotide monomers in the oligonucleotide (e.g.,random or ordered combination of deoxyribonucleotide monomers andribonucleotide monomers). An oligonucleotide can be 4 to 10, 10 to 20,21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100,100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400,or 400-500 nucleotides in length, for example. Oligonucleotides caninclude one or more functional moieties that are attached (e.g.,covalently or non-covalently) to the multimer structure. For example, anoligonucleotide can include one or more detectable labels (e.g., aradioisotope or fluorophore).

(v) Subject

A “subject” is an animal, such as a mammal (e.g., human or a non-humansimian), or avian (e.g., bird), or other organism, such as a plant.Examples of subjects include, but are not limited to, a mammal such as arodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig,goat, cow, cat, dog, primate (i.e. human or non-human primate); a plantsuch as Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola,or soybean; an algae such as Chlamydomonas reinhardtii; a nematode suchas Caenorhabditis elegans; an insect such as Drosophila melanogaster,mosquito, fruit fly, or honey bee; an arachnid such as a spider; a fishsuch as zebrafish; a reptile; an amphibian such as a frog or Xenopuslaevis; a Dictyostelium discoideum; a fungi such as Pneumocystiscarinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae orSchizosaccharomyces pombe; or a Plasmodium falciparum.

(vi) Genome

A “genome” generally refers to genomic information from a subject, whichcan be, for example, at least a portion of, or the entirety of, thesubject's gene-encoded hereditary information. A genome can includecoding regions (e.g., that code for proteins) as well as non-codingregions. A genome can include the sequences of some or all of thesubject's chromosomes. For example, the human genome ordinarily has atotal of 46 chromosomes. The sequences of some or all of these canconstitute the genome.

(vii) Adaptor, Adapter, and Tag

An “adaptor,” an “adapter,” and a “tag” are terms that are usedinterchangeably in this disclosure, and refer to species that can becoupled to a polynucleotide sequence (in a process referred to as“tagging”) using any one of many different techniques including (but notlimited to) ligation, hybridization, and tagmentation. Adaptors can alsobe nucleic acid sequences that add a function, e.g., spacer sequences,primer sequences/sites, barcode sequences, unique molecular identifiersequences.

(viii) Hybridizing, Hybridize, Annealing, and Anneal

The terms “hybridizing,” “hybridize,” “annealing,” and “anneal” are usedinterchangeably in this disclosure, and refer to the pairing ofsubstantially complementary or complementary nucleic acid sequenceswithin two different molecules. Pairing can be achieved by any processin which a nucleic acid sequence joins with a substantially or fullycomplementary sequence through base pairing to form a hybridizationcomplex. For purposes of hybridization, two nucleic acid sequences are“substantially complementary” if at least 60% (e.g., at least 70%, atleast 80%, or at least 90%) of their individual bases are complementaryto one another.

(ix) Primer

A “primer” is a single-stranded nucleic acid sequence having a 3′ endthat can be used as a chemical substrate for a nucleic acid polymerasein a nucleic acid extension reaction. RNA primers are formed of RNAnucleotides, and are used in RNA synthesis, while DNA primers are formedof DNA nucleotides and used in DNA synthesis. Primers can also includeboth RNA nucleotides and DNA nucleotides (e.g., in a random or designedpattern). Primers can also include other natural or syntheticnucleotides described herein that can have additional functionality. Insome examples, DNA primers can be used to prime RNA synthesis and viceversa (e.g., RNA primers can be used to prime DNA synthesis). Primerscan vary in length. For example, primers can be about 6 bases to about120 bases. For example, primers can include up to about 25 bases.

(x) Primer Extension

A “primer extension” refers to any method where two nucleic acidsequences (e.g., a constant region from each of two distinct captureprobes) become linked (e.g., hybridized) by an overlap of theirrespective terminal complementary nucleic acid sequences (i.e., forexample, 3′ termini). Such linking can be followed by nucleic acidextension (e.g., an enzymatic extension) of one, or both termini usingthe other nucleic acid sequence as a template for extension. Enzymaticextension can be performed by an enzyme including, but not limited to, apolymerase and/or a reverse transcriptase.

(xi) Proximity Ligation

A “proximity ligation” is a method of ligating two (or more) nucleicacid sequences that are in proximity with each other through enzymaticmeans (e.g., a ligase). In some embodiments, proximity ligation caninclude a “gap-filling” step that involves incorporation of one or morenucleic acids by a polymerase, based on the nucleic acid sequence of atemplate nucleic acid molecule, spanning a distance between the twonucleic acid molecules of interest (see, e.g., U.S. Pat. No. 7,264,929,the entire contents of which are incorporated herein by reference).

A wide variety of different methods can be used for proximity ligatingnucleic acid molecules, including (but not limited to) “sticky-end” and“blunt-end” ligations. Additionally, single-stranded ligation can beused to perform proximity ligation on a single-stranded nucleic acidmolecule. Sticky-end proximity ligations involve the hybridization ofcomplementary single-stranded sequences between the two nucleic acidmolecules to be joined, prior to the ligation event itself. Blunt-endproximity ligations generally do not include hybridization ofcomplementary regions from each nucleic acid molecule because bothnucleic acid molecules lack a single-stranded overhang at the site ofligation.

(xii) Nucleic Acid Extension

A “nucleic acid extension” generally involves incorporation of one ormore nucleic acids (e.g., A, G, C, T, U, nucleotide analogs, orderivatives thereof) into a molecule (such as, but not limited to, anucleic acid sequence) in a template-dependent manner, such thatconsecutive nucleic acids are incorporated by an enzyme (such as apolymerase or reverse transcriptase), thereby generating a newlysynthesized nucleic acid molecule. For example, a primer that hybridizesto a complementary nucleic acid sequence can be used to synthesize a newnucleic acid molecule by using the complementary nucleic acid sequenceas a template for nucleic acid synthesis. Similarly, a 3′ polyadenylatedtail of an mRNA transcript that hybridizes to a poly (dT) sequence(e.g., capture domain) can be used as a template for single-strandsynthesis of a corresponding cDNA molecule.

(xiii) PCR Amplification

A “PCR amplification” refers to the use of a polymerase chain reaction(PCR) to generate copies of genetic material, including DNA and RNAsequences. Suitable reagents and conditions for implementing PCR aredescribed, for example, in U.S. Pat. Nos. 4,683,202, 4,683,195,4,800,159, 4,965,188, and 5,512,462, the entire contents of each ofwhich are incorporated herein by reference. In a typical PCRamplification, the reaction mixture includes the genetic material to beamplified, an enzyme, one or more primers that are employed in a primerextension reaction, and reagents for the reaction. The oligonucleotideprimers are of sufficient length to provide for hybridization tocomplementary genetic material under annealing conditions. The length ofthe primers generally depends on the length of the amplificationdomains, but will typically be at least 4 bases, at least 5 bases, atleast 6 bases, at least 8 bases, at least 9 bases, at least 10 basepairs (bp), at least 11 bp, at least 12 bp, at least 13 bp, at least 14bp, at least 15 bp, at least 16 bp, at least 17 bp, at least 18 bp, atleast 19 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35bp, and can be as long as 40 bp or longer, where the length of theprimers will generally range from 18 to 50 bp. The genetic material canbe contacted with a single primer or a set of two primers (forward andreverse primers), depending upon whether primer extension, linear orexponential amplification of the genetic material is desired.

In some embodiments, the PCR amplification process uses a DNA polymeraseenzyme. The DNA polymerase activity can be provided by one or moredistinct DNA polymerase enzymes. In certain embodiments, the DNApolymerase enzyme is from a bacterium, e.g., the DNA polymerase enzymeis a bacterial DNA polymerase enzyme. For instance, the DNA polymerasecan be from a bacterium of the genus Escherichia, Bacillus, Thermus, orPyrococcus.

Suitable examples of DNA polymerases that can be used include, but arenot limited to: E. coli DNA polymerase I, Bsu DNA polymerase, Bst DNApolymerase, Taq DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNApolymerase, LongAmp® Taq DNA polymerase, LongAmp® Hot Start Taq DNApolymerase, Crimson LongAmp® Taq DNA polymerase, Crimson Taq DNApolymerase, OneTaq® DNA polymerase, OneTaq® Quick-Load® DNA polymerase,Hemo KlenTaq® DNA polymerase, REDTaq® DNA polymerase, Phusion® DNApolymerase, Phusion® High-Fidelity DNA polymerase, Platinum Pfx DNApolymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, Klenowfragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase andT7 DNA polymerase enzymes.

The term “DNA polymerase” includes not only naturally-occurring enzymesbut also all modified derivatives thereof, including derivatives ofnaturally-occurring DNA polymerase enzymes. For instance, in someembodiments, the DNA polymerase is modified to remove 5′-3′ exonucleaseactivity. Sequence-modified derivatives or mutants of DNA polymeraseenzymes that can be used include, but are not limited to, mutants thatretain at least some of the functional, e.g., DNA polymerase activity ofthe wild-type sequence. Mutations can affect the activity profile of theenzymes, e.g., enhance or reduce the rate of polymerization, underdifferent reaction conditions, e.g., temperature, templateconcentration, primer concentration, etc. Mutations orsequence-modifications can also affect the exonuclease activity and/orthermostability of the enzyme.

In some embodiments, PCR amplification can include reactions such as,but not limited to, a strand-displacement amplification reaction, arolling circle amplification reaction, a ligase chain reaction, atranscription-mediated amplification reaction, an isothermalamplification reaction, and/or a loop-mediated amplification reaction.

In some embodiments, PCR amplification uses a single primer that iscomplementary to the 3′ tag of target DNA fragments. In someembodiments, PCR amplification uses a first and a second primer, whereat least a 3′ end portion of the first primer is complementary to atleast a portion of the 3′ tag of the target nucleic acid fragments, andwhere at least a 3′ end portion of the second primer exhibits thesequence of at least a portion of the 5′ tag of the target nucleic acidfragments. In some embodiments, a 5′ end portion of the first primer isnon-complementary to the 3′ tag of the target nucleic acid fragments,and a 5′ end portion of the second primer does not exhibit the sequenceof at least a portion of the 5′ tag of the target nucleic acidfragments. In some embodiments, the first primer includes a firstuniversal sequence and/or the second primer includes a second universalsequence.

In some embodiments (e.g., when the PCR amplification amplifies capturedDNA), the PCR amplification products can be ligated to additionalsequences using a DNA ligase enzyme. The DNA ligase activity can beprovided by one or more distinct DNA ligase enzymes. In someembodiments, the DNA ligase enzyme is from a bacterium, e.g., the DNAligase enzyme is a bacterial DNA ligase enzyme. In some embodiments, theDNA ligase enzyme is from a virus (e.g., a bacteriophage). For instance,the DNA ligase can be T4 DNA ligase. Other enzymes appropriate for theligation step include, but are not limited to, Tth DNA ligase, Taq DNAligase, Thermococcus sp. (strain 9oN) DNA ligase (9oN™ DNA ligase,available from New England Biolabs, Ipswich, Mass.), and Ampligase®(available from Lucigen, Middleton, Wis.). Derivatives, e.g.,sequence-modified derivatives, and/or mutants thereof, can also be used.

In some embodiments, genetic material is amplified by reversetranscription polymerase chain reaction (RT-PCR). The desired reversetranscriptase activity can be provided by one or more distinct reversetranscriptase enzymes (i.e., RNA dependent DNA polymerases), suitableexamples of which include, but are not limited to: M-MLV, MuLV, AMV,HIV, ArrayScript™, MultiScribe™, ThermoScript™, and SuperScript® I, II,III, and IV enzymes. “Reverse transcriptase” includes not only naturallyoccurring enzymes, but all such modified derivatives thereof, includingalso derivatives of naturally-occurring reverse transcriptase enzymes.

In addition, reverse transcription can be performed usingsequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIVreverse transcriptase enzymes, including mutants that retain at leastsome of the functional, e.g., reverse transcriptase, activity of thewild-type sequence. The reverse transcriptase enzyme can be provided aspart of a composition that includes other components, e.g., stabilizingcomponents that enhance or improve the activity of the reversetranscriptase enzyme, such as RNase inhibitor(s), inhibitors ofDNA-dependent DNA synthesis, e.g., actinomycin D. Many sequence-modifiedderivative or mutants of reverse transcriptase enzymes, e.g., M-MLV, andcompositions including unmodified and modified enzymes are commerciallyavailable, e.g., ArrayScript™, MultiScribe™, ThermoScript™, andSuperScript® I, II, III, and IV enzymes.

Certain reverse transcriptase enzymes (e.g., Avian Myeloblastosis Virus(AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV,MMLV) Reverse Transcriptase) can synthesize a complementary DNA strandusing both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as atemplate. Thus, in some embodiments, the reverse transcription reactioncan use an enzyme (reverse transcriptase) that is capable of using bothRNA and ssDNA as the template for an extension reaction, e.g., an AMV orMMLV reverse transcriptase.

In some embodiments, the quantification of RNA and/or DNA is carried outby real-time PCR (also known as quantitative PCR or qPCR), usingtechniques well known in the art, such as but not limited to “TAQMAN™”,or dyes such as “SYBR®”, or on capillaries (“LightCycler® Capillaries”).In some embodiments, the quantification of genetic material isdetermined by optical absorbance and with real-time PCR. In someembodiments, the quantification of genetic material is determined bydigital PCR. In some embodiments, the genes analyzed can be compared toa reference nucleic acid extract (DNA and RNA) corresponding to theexpression (mRNA) and quantity (DNA) in order to compare expressionlevels of the target nucleic acids.

(xiv) Antibody

An “antibody” is a polypeptide molecule that recognizes and binds to acomplementary target antigen. Antibodies typically have a molecularstructure shape that resembles a Y shape, or polymers thereof.Naturally-occurring antibodies, referred to as immunoglobulins, belongto one of the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE.Antibodies can also be produced synthetically. For example, recombinantantibodies, which are monoclonal antibodies, can be synthesized usingsynthetic genes by recovering the antibody genes from source cells,amplifying into an appropriate vector, and introducing the vector into ahost to cause the host to express the recombinant antibody. In general,recombinant antibodies can be cloned from any species ofantibody-producing animal using suitable oligonucleotide primers and/orhybridization probes. Recombinant techniques can be used to generateantibodies and antibody fragments, including non-endogenous species.

Synthetic antibodies can be derived from non-immunoglobulin sources. Forexample, antibodies can be generated from nucleic acids (e.g.,aptamers), and from non-immunoglobulin protein scaffolds (such aspeptide aptamers) into which hypervariable loops are inserted to formantigen binding sites. Synthetic antibodies based on nucleic acids orpeptide structures can be smaller than immunoglobulin-derivedantibodies, leading to greater tissue penetration.

Antibodies can also include affimer proteins, which are affinityreagents that typically have a molecular weight of about 12-14 kDa.Affimer proteins generally bind to a target (e.g., a target protein)with both high affinity and specificity. Examples of such targetsinclude, but are not limited to, ubiquitin chains, immunoglobulins, andC-reactive protein. In some embodiments, affimer proteins are derivedfrom cysteine protease inhibitors, and include peptide loops and avariable N-terminal sequence that provides the binding site.

Antibodies can also include single domain antibodies (VHH domains andVNAR domains), scFvs, and Fab fragments.

(xv) Affinity Group

An “affinity group” is a molecule or molecular moiety which has a highaffinity or preference for associating or binding with another specificor particular molecule or moiety. The association or binding withanother specific or particular molecule or moiety can be via anon-covalent interaction, such as hydrogen bonding, ionic forces, andvan der Waals interactions. An affinity group can, for example, bebiotin, which has a high affinity or preference to associate or bind tothe protein avidin or streptavidin. An affinity group, for example, canalso refer to avidin or streptavidin which has an affinity to biotin.Other examples of an affinity group and specific or particular moleculeor moiety to which it binds or associates with include, but are notlimited to, antibodies or antibody fragments and their respectiveantigens, such as digoxigenin and anti-digoxigenin antibodies, lectin,and carbohydrates (e.g., a sugar, a monosaccharide, a disaccharide, or apolysaccharide), and receptors and receptor ligands.

Any pair of affinity group and its specific or particular molecule ormoiety to which it binds or associates with can have their rolesreversed, for example, such that between a first molecule and a secondmolecule, in a first instance the first molecule is characterized as anaffinity group for the second molecule, and in a second instance thesecond molecule is characterized as an affinity group for the firstmolecule.

(xvi) Label, Detectable Label, and Optical Label

The terms “detectable label,” “optical label,” and “label” are usedinterchangeably herein to refer to a directly or indirectly detectablemoiety that is associated with (e.g., conjugated to) a molecule to bedetected, e.g., a capture probe or analyte. The detectable label can bedirectly detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, can be indirectlydetectable, e.g., by catalyzing chemical alterations of a chemicalsubstrate compound or composition, which chemical substrate compound orcomposition is directly detectable. Detectable labels can be suitablefor small scale detection and/or suitable for high-throughput screening.As such, suitable detectable labels include, but are not limited to,radioisotopes, fluorophores, chemiluminescent compounds, bioluminescentcompounds, and dyes.

The detectable label can be qualitatively detected (e.g., optically orspectrally), or it can be quantified. Qualitative detection generallyincludes a detection method in which the existence or presence of thedetectable label is confirmed, whereas quantifiable detection generallyincludes a detection method having a quantifiable (e.g., numericallyreportable) value such as an intensity, duration, polarization, and/orother properties. In some embodiments, the detectable label is bound toa feature or to a capture probe associated with a feature. For example,detectably labeled features can include a fluorescent, a colorimetric,or a chemiluminescent label attached to a bead (see, for example,Rajeswari et al., J. Microbiol Methods 139:22-28, 2017, and Forcucci etal., J. Biomed Opt. 10:105010, 2015, the entire contents of each ofwhich are incorporated herein by reference).

In some embodiments, a plurality of detectable labels can be attached toa feature, capture probe, or composition to be detected. For example,detectable labels can be incorporated during nucleic acid polymerizationor amplification (e.g., Cy5®-labelled nucleotides, such as Cy5®-dCTP).Any suitable detectable label can be used. In some embodiments, thedetectable label is a fluorophore. For example, the fluorophore can befrom a group that includes: 7-AAD (7-Aminoactinomycin D), AcridineOrange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350, Alexa Fluor®430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor®555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor®647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor®750, Allophycocyanin (APC), AMCA/AMCA-X, 7-Aminoactinomycin D (7-AAD),7-Amino-4-methylcoumarin, 6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7,ATTO-TAG™ CBQCA, ATTO-TAG™ FQ, Auramine O-Feulgen, BCECF (high pH), BFP(Blue Fluorescent Protein), BFP/GFP FRET, BOBO™-1/BO-PRO™-1,BOBO™-3/BO-PRO™-3, BODIPY® FL, BODIPY® TMR, BODIPY® TR-X, BODIPY®530/550, BODIPY® 558/568, BODIPY® 564/570, BODIPY® 581/591, BODIPY®630/650-X, BODIPY® 650-665-X, BTC, Calcein, Calcein Blue, CalciumCrimson™, Calcium Green-1™, Calcium Orange™, Calcofluor® White,5-Carboxyfluoroscein (5-FAM), 5-Carboxynaphthofluoroscein,6-Carboxyrhodamine 6G, 5-Carboxytetramethylrhodamine (5-TAMRA),Carboxy-X-rhodamine (5-ROX), Cascade Blue®, Cascade Yellow™, CCF2(GeneBLAzer™), CFP (Cyan Fluorescent Protein), CFP/YFP FRET, ChromomycinA3, C1-NERF (low pH), CPM, 6-CR 6G, CTC Formazan, Cy2®, Cy3®, Cy3.5®,Cy5®, Cy5.5®, Cy7®, Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine,Dansylchloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD(DilC18(5)), DIDS, Dil (DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)),Di-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (Red FluorescentProtein), EBFP, ECFP, EGFP, ELF®-97 alcohol, Eosin, Erythrosin, Ethidiumbromide, Ethidium homodimer-1 (EthD-1), Europium (III) Chloride, 5-FAM(5-Carboxyfluorescein), Fast Blue, Fluorescein-dT phosphoramidite, FITC,Fluo-3, Fluo-4, FluorX®, Fluoro-Gold™ (high pH), Fluoro-Gold™ (low pH),Fluoro-Jade, FM® 1-43, Fura-2 (high calcium), Fura-2/BCECF, Fura Red™(high calcium), Fura Red™/Fluo-3, GeneBLAzer™ (CCF2), GFP Red Shifted(rsGFP), GFP Wild Type, GFP/BFP FRET, GFP/DsRed FRET, Hoechst 33342 &33258, 7-Hydroxy-4-methylcoumarin (pH 9), 1,5 IAEDANS, Indo-1 (highcalcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine,JC-1, 6-JOE, JOJO™-1/JO-PRO™-1, LDS 751 (+DNA), LDS 751 (+RNA),LOLO™-1/LO-PRO™-1, Lucifer Yellow, LysoSensor™ Blue (pH 5), LysoSensor™Green (pH 5), LysoSensor™ Yellow/Blue (pH 4.2), LysoTracker® Green,LysoTracker® Red, LysoTracker® Yellow, Mag-Fura-2, Mag-Indo-1, MagnesiumGreen™ Marina Blue®, 4-Methylumbelliferone, Mithramycin, MitoTracker®Green, MitoTracker® Orange, MitoTracker® Red, NBD (amine), Nile Red,Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue,PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP(Peridinin chlorphyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7),C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (PropidiumIodide), PKH26, PKH67, POPO™-1/PO-PRO™-1, POPO™-3/PO-PRO™-3, PropidiumIodide (PI), PyMPO, Pyrene, Pyronin Y, Quantam Red (PE-Cy5), QuinacrineMustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red Fluorescent Protein(DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine Green™,Rhodamine Red™, Rhodamine Phalloidin, Rhodamine 110, Rhodamine 123,5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS,SNAFL®-1 (high pH), SNAFL®-2, SNARF®-1 (high pH), SNARF®-1 (low pH),Sodium Green™, SpectrumAqua®, SpectrumGreen® #1, SpectrumGreen® #2,SpectrumOrange®, SpectrumRed®, SYTO® 11, SYTO® 13, SYTO® 17, SYTO® 45,SYTOX® Blue, SYTOX® Green, SYTOX® Orange, 5-TAMRA(5-Carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), TexasRed®/Texas Red®-X, Texas Red®-X (NHS Ester), Thiadicarbocyanine,Thiazole Orange, TOTO®-1/TO-PRO®-1, TOTO®-3/TO-PRO®-3, TO-PRO®-5,Tri-color (PE-Cy5), TRITC (Tetramethylrhodamine), TruRed (PerCP-Cy5.5),WW 781, X-Rhodamine (XRITC), Y66F, Y66H, Y66 W, YFP (Yellow FluorescentProtein), YOYO®-1/YO-PRO®-1, YOYO®-3/YO-PRO®-3, 6-FAM (Fluorescein),6-FAM (NHS Ester), 6-FAM (Azide), HEX, TAMRA (NHS Ester), Yakima Yellow,MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101,ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5′ IRDye® 700,5′ IRDye® 800, 5′ IRDye® 800CW (NHS Ester), WellRED D4 Dye, WellRED D3Dye, WellRED D2 Dye, Lightcycler® 640 (NHS Ester), and Dy 750 (NHSEster).

As mentioned above, in some embodiments, a detectable label is orincludes a luminescent or chemiluminescent moiety. Commonluminescent/chemiluminescent moieties include, but are not limited to,peroxidases such as horseradish peroxidase (HRP), soybean peroxidase(SP), alkaline phosphatase, and luciferase. These protein moieties cancatalyze chemiluminescent reactions given the appropriate chemicalsubstrates (e.g., an oxidizing reagent plus a chemiluminescentcompound). A number of compound families are known to providechemiluminescence under a variety of conditions. Non-limiting examplesof chemiluminescent compound families include2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- andthe dimethylamino[ca]benz analog. These compounds can luminesce in thepresence of alkaline hydrogen peroxide or calcium hypochlorite and base.Other examples of chemiluminescent compound families include, e.g.,2,4,5-triphenylimidazoles, para-dimethylamino and -methoxy substituents,oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinumesters, luciferins, lucigenins, or acridinium esters.

(xvii) Template Switching Oligonucleotide

A “template switching oligonucleotide” is an oligonucleotide thathybridizes to untemplated nucleotides added by a reverse transcriptase(e.g., enzyme with terminal transferase activity) during reversetranscription. In some embodiments, a template switching oligonucleotidehybridizes to untemplated poly(C) nucleotides added by a reversetranscriptase. In some embodiments, the template switchingoligonucleotide adds a common 5′ sequence to full-length cDNA that isused for cDNA amplification.

In some embodiments, the template switching oligonucleotide adds acommon sequence onto the 5′ end of the RNA being reverse transcribed.For example, a template switching oligonucleotide can hybridize tountemplated poly(C) nucleotides added onto the end of a cDNA moleculeand provide a template for the reverse transcriptase to continuereplication to the 5′ end of the template switching oligonucleotide,thereby generating full-length cDNA ready for further amplification. Insome embodiments, once a full-length cDNA molecule is generated, thetemplate switching oligonucleotide can serve as a primer in a cDNAamplification reaction.

In some embodiments, a template switching oligonucleotide is addedbefore, contemporaneously with, or after a reverse transcription, orother terminal transferase-based reaction. In some embodiments, atemplate switching oligonucleotide is included in the capture probe. Incertain embodiments, methods of sample analysis using template switchingoligonucleotides can involve the generation of nucleic acid productsfrom analytes of the tissue sample, followed by further processing ofthe nucleic acid products with the template switching oligonucleotide.

Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target. In some embodiments, thehybridization region can, e.g., include a series of G bases tocomplement the overhanging C bases at the 3′ end of a cDNA molecule. Theseries of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases,5 G bases, or more than 5 G bases. The template sequence can include anysequence to be incorporated into the cDNA. In other embodiments, thehybridization region can include at least one base in addition to atleast one G base. In other embodiments, the hybridization can includebases that are not a G base. In some embodiments, the template regionincludes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequencesand/or functional sequences. In some embodiments, the template regionand hybridization region are separated by a spacer.

In some embodiments, the template regions include a barcode sequence.The barcode sequence can act as a spatial barcode and/or as a uniquemolecular identifier. Template switching oligonucleotides can includedeoxyribonucleic acids; ribonucleic acids; modified nucleic acidsincluding 2-aminopurine, 2,6-diaminopurine (2-amino-dA), inverted dT,5-methyl dC, 2′-deoxylnosine, Super T (5-hydroxybutynl-2′-deoxyuridine),Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlockednucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC,2′ fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), orany combination of the foregoing.

In some embodiments, the length of a template switching oligonucleotidecan be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250nucleotides or longer. In some embodiments, the length of a templateswitching oligonucleotide can be at most about 2, 10, 20, 50, 100, 150,200, or 250 nucleotides or longer.

(xviii) Splint Oligonucleotide

A “splint oligonucleotide” is an oligonucleotide that, when hybridizedto other polynucleotides, acts as a “splint” to position thepolynucleotides next to one another so that they can be ligatedtogether. In some embodiments, the splint oligonucleotide is DNA or RNA.The splint oligonucleotide can include a nucleotide sequence that ispartially complimentary to nucleotide sequences from two or moredifferent oligonucleotides. In some embodiments, the splintoligonucleotide assists in ligating a “donor” oligonucleotide and an“acceptor” oligonucleotide. In general, an RNA ligase, a DNA ligase, oranother other variety of ligase is used to ligate two nucleotidesequences together

In some embodiments, the splint oligonucleotide is between 10 and 50oligonucleotides in length, e.g., between 10 and 45, 10 and 40, 10 and35, 10 and 30, 10 and 25, or 10 and 20 oligonucleotides in length. Insome embodiments, the splint oligonucleotide is between 15 and 50, 15and 45, 15 and 40, 15 and 35, 15 and 30, 15 and 30, or 15 and 25nucleotides in length.

(c) Analytes

The apparatus, systems, methods, and compositions described in thisdisclosure can be used to detect and analyze a wide variety of differentanalytes. For the purpose of this disclosure, an “analyte” can includeany biological substance, structure, moiety, or component to beanalyzed. The term “target” can similarly refer to an analyte ofinterest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral coat proteins,extracellular and intracellular proteins, antibodies, and antigenbinding fragments. In some embodiments, the analyte can be an organelle(e.g., nuclei or mitochondria).

Cell surface features corresponding to analytes can include, but are notlimited to, a receptor, an antigen, a surface protein, a transmembraneprotein, a cluster of differentiation protein, a protein channel, aprotein pump, a carrier protein, a phospholipid, a glycoprotein, aglycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, an extracellular matrix protein, aposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation)state of a cell surface protein, a gap junction, and an adherensjunction.

Analytes can be derived from a specific type of cell and/or a specificsub-cellular region. For example, analytes can be derived from cytosol,from cell nuclei, from mitochondria, from microsomes, and moregenerally, from any other compartment, organelle, or portion of a cell.Permeabilizing agents that specifically target certain cell compartmentsand organelles can be used to selectively release analytes from cellsfor analysis.

Examples of nucleic acid analytes include DNA analytes such as genomicDNA, methylated DNA, specific methylated DNA sequences, fragmented DNA,mitochondrial DNA, in situ synthesized PCR products, and RNA/DNAhybrids.

Examples of nucleic acid analytes also include RNA analytes such asvarious types of coding and non-coding RNA. Examples of the differenttypes of RNA analytes include messenger RNA (mRNA), ribosomal RNA(rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNAcan be a transcript (e.g., present in a tissue section). The RNA can besmall (e.g., less than 200 nucleic acid bases in length) or large (e.g.,RNA greater than 200 nucleic acid bases in length). Small RNAs mainlyinclude 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA),microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA(snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA),and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNAor single-stranded RNA. The RNA can be circular RNA. The RNA can be abacterial rRNA (e.g., 16s rRNA or 23s rRNA).

Additional examples of analytes include mRNA and cell surface features(e.g., using the labelling agents described herein), mRNA andintracellular proteins (e.g., transcription factors), mRNA and cellmethylation status, mRNA and accessible chromatin (e.g., ATAC-seq,DNase-seq, and/or MNase-seq), mRNA and metabolites (e.g., using thelabelling agents described herein), a barcoded labelling agent (e.g.,the oligonucleotide tagged antibodies described herein) and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), mRNA and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein). In someembodiments, a perturbation agent can be a small molecule, an antibody,a drug, an aptamer, a miRNA, a physical environmental (e.g., temperaturechange), or any other known perturbation agents.

Analytes can include a nucleic acid molecule with a nucleic acidsequence encoding at least a portion of a V(D)J sequence of an immunecell receptor (e.g., a TCR or BCR). In some embodiments, the nucleicacid molecule is cDNA first generated from reverse transcription of thecorresponding mRNA, using a poly(T) containing primer. The generatedcDNA can then be barcoded using a capture probe, featuring a barcodesequence (and optionally, a UMI sequence) that hybridizes with at leasta portion of the generated cDNA. In some embodiments, a templateswitching oligonucleotide hybridizes to a poly(C) tail added to a 3′ endof the cDNA by a reverse transcriptase enzyme. The original mRNAtemplate and template switching oligonucleotide can then be denaturedfrom the cDNA and the barcoded capture probe can then hybridize with thecDNA and a complement of the cDNA generated. Additional methods andcompositions suitable for barcoding cDNA generated from mRNA transcriptsincluding those encoding V(D)J regions of an immune cell receptor and/orbarcoding methods and composition including a template switcholigonucleotide are described in PCT Patent ApplicationPCT/US2017/057269, filed Oct. 18, 2017, and U.S. patent application Ser.No. 15/825,740, filed Nov. 29, 2017, both of which are incorporatedherein by reference in their entireties. V(D)J analysis can also becompleted with the use of one or more labelling agents that bind toparticular surface features of immune cells and associated with barcodesequences. The one or more labelling agents can include an MHC or MHCmultimer.

As described above, the analyte can include a nucleic acid capable offunctioning as a component of a gene editing reaction, such as, forexample, clustered regularly interspaced short palindromic repeats(CRISPR)-based gene editing. Accordingly, the capture probe can includea nucleic acid sequence that is complementary to the analyte (e.g., asequence that can hybridize to the CRISPR RNA (crRNA), single guide RNA(sgRNA), or an adapter sequence engineered into a crRNA or sgRNA).

In certain embodiments, an analyte can be extracted from a live cell.Processing conditions can be adjusted to ensure that a biological sampleremains live during analysis, and analytes are extracted from (orreleased from) live cells of the sample. Live cell-derived analytes canbe obtained only once from the sample, or can be obtained at intervalsfrom a sample that continues to remain in viable condition.

In general, the systems, apparatus, methods, and compositions can beused to analyze any number of analytes. For example, the number ofanalytes that are analyzed can be at least about 2, at least about 3, atleast about 4, at least about 5, at least about 6, at least about 7, atleast about 8, at least about 9, at least about 10, at least about 11,at least about 12, at least about 13, at least about 14, at least about15, at least about 20, at least about 25, at least about 30, at leastabout 40, at least about 50, at least about 100, at least about 1,000,at least about 10,000, at least about 100,000 or more different analytespresent in a region of the sample or within an individual feature of thesubstrate. Methods for performing multiplexed assays to analyze two ormore different analytes will be discussed in a subsequent section ofthis disclosure.

(d) Biological Samples

(i) Types of Biological Samples

A “biological sample” is obtained from the subject for analysis usingany of a variety of techniques including, but not limited to, biopsy,surgery, and laser capture microscopy (LCM), and generally includescells and/or other biological material from the subject. In addition tothe subjects described above, a biological sample can be obtained fromnon-mammalian organisms (e.g., a plants, an insect, an arachnid, anematode (e.g., Caenorhabditis elegans), a fungi, an amphibian, or afish (e.g., zebrafish)). A biological sample can be obtained from aprokaryote such as a bacterium, e.g., Escherichia coli, Staphylococci orMycoplasma pneumoniae; an archaea; a virus such as Hepatitis C virus orhuman immunodeficiency virus; or a viroid. A biological sample can beobtained from a eukaryote, such as a patient derived organoid (PDO) orpatient derived xenograft (PDX). The biological sample can includeorganoids, a miniaturized and simplified version of an organ produced invitro in three dimensions that shows realistic micro-anatomy. Organoidscan be generated from one or more cells from a tissue, embryonic stemcells, and/or induced pluripotent stem cells, which can self-organize inthree-dimensional culture owing to their self-renewal anddifferentiation capacities. In some embodiments, an organoid is acerebral organoid, an intestinal organoid, a stomach organoid, a lingualorganoid, a thyroid organoid, a thymic organoid, a testicular organoid,a hepatic organoid, a pancreatic organoid, an epithelial organoid, alung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or aretinal organoid. Subjects from which biological samples can be obtainedcan be healthy or asymptomatic individuals, individuals that have or aresuspected of having a disease (e.g., cancer) or a pre-disposition to adisease, and/or individuals that are in need of therapy or suspected ofneeding therapy.

Biological samples can be derived from a homogeneous culture orpopulation of the subjects or organisms mentioned herein oralternatively from a collection of several different organisms, forexample, in a community or ecosystem.

Biological samples can include one or more diseased cells. A diseasedcell can have altered metabolic properties, gene expression, proteinexpression, and/or morphologic features. Examples of diseases includeinflammatory disorders, metabolic disorders, nervous system disorders,and cancer. Cancer cells can be derived from solid tumors, hematologicalmalignancies, cell lines, or obtained as circulating tumor cells.

Biological samples can also include fetal cells. For example, aprocedure such as amniocentesis can be performed to obtain a fetal cellsample from maternal circulation. Sequencing of fetal cells can be usedto identify any of a number of genetic disorders, including, e.g.,aneuploidy such as Down's syndrome, Edwards syndrome, and Patausyndrome. Further, cell surface features of fetal cells can be used toidentify any of a number of disorders or diseases.

Biological samples can also include immune cells. Sequence analysis ofthe immune repertoire of such cells, including genomic, proteomic, andcell surface features, can provide a wealth of information to facilitatean understanding the status and function of the immune system. By way ofexample, determining the status (e.g., negative or positive) of minimalresidue disease (MRD) in a multiple myeloma (MM) patient followingautologous stem cell transplantation is considered a predictor of MRD inthe MM patient (see, e.g., U.S. Patent Application Publication No.2018/0156784, the entire contents of which are incorporated herein byreference).

Examples of immune cells in a biological sample include, but are notlimited to, B cells, T cells (e.g., cytotoxic T cells, natural killer Tcells, regulatory T cells, and T helper cells), natural killer cells,cytokine induced killer (CIK) cells, myeloid cells, such as granulocytes(basophil granulocytes, eosinophil granulocytes, neutrophilgranulocytes/hypersegmented neutrophils), monocytes/macrophages, mastcells, thrombocytes/megakaryocytes, and dendritic cells.

The biological sample can include any number of macromolecules, forexample, cellular macromolecules and organelles (e.g., mitochondria andnuclei). The biological sample can be a nucleic acid sample and/orprotein sample. The biological sample can be a carbohydrate sample or alipid sample. The biological sample can be obtained as a tissue sample,such as a tissue section, biopsy, a core biopsy, needle aspirate, orfine needle aspirate. The sample can be a fluid sample, such as a bloodsample, urine sample, or saliva sample. The sample can be a skin sample,a colon sample, a cheek swab, a histology sample, a histopathologysample, a plasma or serum sample, a tumor sample, living cells, culturedcells, a clinical sample such as, for example, whole blood orblood-derived products, blood cells, or cultured tissues or cells,including cell suspensions.

Cell-free biological samples can include extracellular polynucleotides.Extracellular polynucleotides can be isolated from a bodily sample,e.g., blood, plasma, serum, urine, saliva, mucosal excretions, sputum,stool, and tears.

As discussed above, a biological sample can include a single analyte ofinterest, or more than one analyte of interest. Methods for performingmultiplexed assays to analyze two or more different analytes in a singlebiological sample is discussed in a subsequent section of thisdisclosure.

(ii) Preparation of Biological Samples

A variety of steps can be performed to prepare a biological sample foranalysis.

Except where indicated otherwise, the preparative steps described belowcan generally be combined in any manner to appropriately prepare aparticular sample for analysis.

(1) Tissue Sectioning

A biological sample can be harvested from a subject (e.g., via surgicalbiopsy, whole subject sectioning), grown in vitro on a growth substrateor culture dish as a population of cells, or prepared as a tissue sliceor tissue section. Grown samples may be sufficiently thin for analysiswithout further processing steps. Alternatively, grown samples, andsamples obtained via biopsy or sectioning, can be prepared as thintissue sections using a mechanical cutting apparatus such as a vibratingblade microtome. As another alternative, in some embodiments, a thintissue section can be prepared by applying a touch imprint of abiological sample to a suitable substrate material.

The thickness of the tissue section can be a fraction of (e.g., lessthan 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximumcross-sectional dimension of a cell. However, tissue sections having athickness that is larger than the maximum cross-section cell dimensioncan also be used. For example, cryostat sections can be used, which canbe, e.g., 10-20 micrometers thick.

More generally, the thickness of a tissue section typically depends onthe method used to prepare the section and the physical characteristicsof the tissue, and therefore sections having a wide variety of differentthicknesses can be prepared and used. For example, the thickness of thetissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50micrometers. Thicker sections can also be used if desired or convenient,e.g., at least 70, 80, 90, or 100 micrometers or more. Typically, thethickness of a tissue section is between 1-100 micrometers, 1-50micrometers, 1-30 micrometers, 1-25 micrometers, 1-20 micrometers, 1-15micrometers, 1-10 micrometers, 2-8 micrometers, 3-7 micrometers, or 4-6micrometers, but as mentioned above, sections with thicknesses larger orsmaller than these ranges can also be analysed.

Multiple sections can also be obtained from a single biological sample.For example, multiple tissue sections can be obtained from a surgicalbiopsy sample by performing serial sectioning of the biopsy sample usinga sectioning blade. Spatial information among the serial sections can bepreserved in this manner, and the sections can be analysed successivelyto obtain three-dimensional information about the biological sample.

(2) Freezing

In some embodiments, the biological sample (e.g., a tissue section asdescribed above) can be prepared by deep freezing at a temperaturesuitable to maintain or preserve the integrity (e.g., the physicalcharacteristics) of the tissue structure. Such a temperature can be,e.g., less than −20° C., or less than −25° C., −30° C., −40° C., −50°C., −60° C., −70° C., 80° C. −90° C., −100° C., −110° C., −120° C.,−130° C., −140° C., −150° C., −160° C., −170° C., −180° C., −190° C., or−200° C. The frozen tissue sample can be sectioned, e.g., thinly sliced,onto a substrate surface using any number of suitable methods. Forexample, a tissue sample can be prepared using a chilled microtome(e.g., a cryostat) set at a temperature suitable to maintain both thestructural integrity of the tissue sample and the chemical properties ofthe nucleic acids in the sample. Such a temperature can be, e.g., lessthan −15° C., less than −20° C., or less than −25° C. A sample can besnap frozen in isopentane and liquid nitrogen. Frozen samples can bestored in a sealed container prior to embedding.

(3) Formalin Fixation and Paraffin Embedding

In some embodiments, the biological sample can be prepared usingformalin-fixation and paraffin-embedding (FFPE), which are establishedmethods. In some embodiments, cell suspensions and other non-tissuesamples can be prepared using formalin-fixation and paraffin-embedding.Following fixation of the sample and embedding in a paraffin or resinblock, the sample can be sectioned as described above. Prior toanalysis, the paraffin-embedding material can be removed from the tissuesection (e.g., deparaffinization) by incubating the tissue section in anappropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5%ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2minutes).

(4) Fixation

As an alternative to formalin fixation described above, a biologicalsample can be fixed in any of a variety of other fixatives to preservethe biological structure of the sample prior to analysis. For example, asample can be fixed via immersion in ethanol, methanol, acetone,formaldehyde (e.g., 2% formaldehyde), paraformaldehyde-Triton,glutaraldehyde, or combinations thereof.

In some embodiments, acetone fixation is used with fresh frozen samples,which can include, but are not limited to, cortex tissue, mouseolfactory bulb, human brain tumor, human post-mortem brain, and breastcancer samples. In some embodiments, a compatible fixation method ischosen and/or optimized based on a desired workflow. For example,formaldehyde fixation may be chosen as compatible for workflows usingIHC/IF protocols for protein visualization. As another example, methanolfixation may be chosen for workflows emphasizing RNA/DNA libraryquality. Acetone fixation may be chosen in some applications topermeabilize the tissue. When acetone fixation is performed,pre-permeabilization steps (described below) may not be performed.Alternatively, acetone fixation can be performed in conjunction withpermeabilization steps.

(5) Embedding

As an alternative to paraffin embedding described above, a biologicalsample can be embedded in any of a variety of other embedding materialsto provide a substrate to the sample prior to sectioning and otherhandling steps. In general, the embedding material is removed prior toanalysis of tissue sections obtained from the sample. Suitable embeddingmaterials include, but are not limited to, waxes, resins (e.g.,methacrylate resins), epoxies, and agar.

(6) Staining

To facilitate visualization, biological samples can be stained using awide variety of stains and staining techniques. In some embodiments, asample can be stained using any number of biological stains, includingbut not limited to, acridine orange, Bismarck brown, carmine, coomassieblue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine,hematoxylin, Hoechst stains, iodine, methyl green, methylene blue,neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide,rhodamine, or safranin.

The sample can be stained using known staining techniques, includingCan-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman,Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's,and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining istypically performed after formalin or acetone fixation.

In some embodiments, the biological sample can be stained using adetectable label (e.g., radioisotopes, fluorophores, chemiluminescentcompounds, bioluminescent compounds, and dyes) as described elsewhereherein. In some embodiments, a biological sample is stained using onlyone type of stain or one technique. In some embodiments, stainingincludes biological staining techniques such as H&E staining. In someembodiments, staining includes identifying analytes usingfluorescently-conjugated antibodies. In some embodiments, a biologicalsample is stained using two or more different types of stains, or two ormore different staining techniques. For example, a biological sample canbe prepared by staining and imaging using one technique (e.g., H&Estaining and brightfield imaging), followed by staining and imagingusing another technique (e.g., IHC/IF staining and fluorescencemicroscopy) for the same biological sample.

In some embodiments, biological samples can be destained. Methods ofdestaining or discoloring a biological sample are known in the art, andgenerally depend on the nature of the stain(s) applied to the sample.For example, H&E staining can be destained by washing the sample in HCl,or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid,benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid,succinic acid, salicylic acid, tartaric acid, sulfurous acid,trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid,orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citricacid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid,hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonicacid, hydrogen sulfide, or combinations thereof). In some embodiments,destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g.,HCl). In some embodiments, destaining can include adding HCl to adownstream solution (e.g., permeabilization solution). In someembodiments, destaining can include dissolving an enzyme used in thedisclosed methods (e.g., pepsin) in an acid (e.g., HCl) solution. Insome embodiments, after destaining hematoxylin with an acid, otherreagents can be added to the destaining solution to raise the pH for usein other applications. For example, SDS can be added to an aciddestaining solution in order to raise the pH as compared to the aciddestaining solution alone. As another example, in some embodiments, oneor more immunofluorescence stains are applied to the sample via antibodycoupling. Such stains can be removed using techniques such as cleavageof disulfide linkages via treatment with a reducing agent and detergentwashing, chaotropic salt treatment, treatment with antigen retrievalsolution, and treatment with an acidic glycine buffer. Methods formultiplexed staining and destaining are described, for example, inBolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin etal., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem.2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009;57:899-905, the entire contents of each of which are incorporated hereinby reference.

(7) Hydrogel Embedding

In some embodiments, hydrogel formation occurs within a biologicalsample. In some embodiments, a biological sample (e.g., tissue section)is embedded in a hydrogel. In some embodiments, hydrogel subunits areinfused into the biological sample, and polymerization of the hydrogelis initiated by an external or internal stimulus. A “hydrogel” asdescribed herein can include a cross-linked 3D network of hydrophilicpolymer chains. A “hydrogel subunit” can be a hydrophilic monomer, amolecular precursor, or a polymer that can be polymerized (e.g.,cross-linked) to form a three-dimensional (3D) hydrogel network.

A hydrogel can swell in the presence of water. In some embodiments, ahydrogel comprises a natural material. In some embodiments, a hydrogelincludes a synthetic material. In some embodiments, a hydrogel includesa hybrid material, e.g., the hydrogel material comprises elements ofboth synthetic and natural polymers. Any of the materials used inhydrogels or hydrogels comprising a polypeptide-based material describedherein can be used. Embedding the sample in this manner typicallyinvolves contacting the biological sample with a hydrogel such that thebiological sample becomes surrounded by the hydrogel. For example, thesample can be embedded by contacting the sample with a suitable polymermaterial, and activating the polymer material to form a hydrogel. Insome embodiments, the hydrogel is formed such that the hydrogel isinternalized within the biological sample.

In some embodiments, the biological sample is immobilized in thehydrogel via cross-linking of the polymer material that forms thehydrogel. Cross-linking can be performed chemically and/orphotochemically, or alternatively by any other hydrogel-formation methodknown in the art. For example, the biological sample can be immobilizedin the hydrogel by polyacrylamide crosslinking. Further, analytes of abiological sample can be immobilized in a hydrogel by crosslinking(e.g., polyacrylamide crosslinking).

The composition and application of the hydrogel to a biological sampletypically depends on the nature and preparation of the biological sample(e.g., sectioned, non-sectioned, fresh-frozen tissue, type of fixation).A hydrogel can be any appropriate hydrogel where upon formation of thehydrogel on the biological sample the biological sample becomes anchoredto or embedded in the hydrogel. Non-limiting examples of hydrogels aredescribed herein or are known in the art. As one example, where thebiological sample is a tissue section, the hydrogel can include amonomer solution and an ammonium persulfate (APS)initiator/tetramethylethylenediamine (TEMED) accelerator solution. Asanother example, where the biological sample consists of cells (e.g.,cultured cells or cells disassociated from a tissue sample), the cellscan be incubated with the monomer solution and APS/TEMED solutions. Forcells, hydrogel are formed in compartments, including but not limited todevices used to culture, maintain, or transport the cells. For example,hydrogels can be formed with monomer solution plus APS/TEMED added tothe compartment to a depth ranging from about 0.1 μm to about 5 mm.

In some embodiments, a hydrogel includes a linker that allows anchoringof the biological sample to the hydrogel. In some embodiments, ahydrogel includes linkers that allow anchoring of biological analytes tothe hydrogel. In such cases, the linker can be added to the hydrogelbefore, contemporaneously with, or after hydrogel formation.Non-limiting examples of linkers that anchor nucleic acids to thehydrogel can include 6-((Acryloyl)amino) hexanoic acid (Acryloyl-X SE)(available from ThermoFisher, Waltham, Mass.), Label-IT Amine (availablefrom MirusBio, Madison, Wis.) and Label X (Chen et al., Nat. Methods13:679-684, (2016)).

In some embodiments, functionalization chemistry can be used. In someembodiments, functionalization chemistry includes hydrogel-tissuechemistry (HTC). Any hydrogel-tissue backbone (e.g., synthetic ornative) suitable for HTC can be used for anchoring biologicalmacromolecules and modulating functionalization. Non-limiting examplesof methods using HTC backbone variants include CLARITY, PACT, ExM,SWITCH and ePACT. In some embodiments, hydrogel formation within abiological sample is permanent. For example, biological macromoleculescan permanently adhere to the hydrogel allowing multiple rounds ofinterrogation. In some embodiments, hydrogel formation within abiological sample is reversible.

In some embodiments, additional reagents are added to the hydrogelsubunits before, contemporaneously with, and/or after polymerization.For example, additional reagents can include but are not limited tooligonucleotides (e.g., capture probes), endonucleases to fragment DNA,fragmentation buffer for DNA, DNA polymerase enzymes, dNTPs used toamplify the nucleic acid and to attach the barcode to the amplifiedfragments. Other enzymes can be used, including without limitation, RNApolymerase, transposase, ligase, proteinase K, and DNAse. Additionalreagents can also include reverse transcriptase enzymes, includingenzymes with terminal transferase activity, primers, and switcholigonucleotides. In some embodiments, optical labels are added to thehydrogel subunits before, contemporaneously with, and/or afterpolymerization.

In some embodiments, HTC reagents are added to the hydrogel before,contemporaneously with, and/or after polymerization. In someembodiments, a cell tagging agent is added to the hydrogel before,contemporaneously with, and/or after polymerization. In someembodiments, a cell-penetrating agent is added to the hydrogel before,contemporaneously with, and/or after polymerization.

In some embodiments, a biological sample is embedded in a hydrogel tofacilitate sample transfer to another location (e.g., to an array). Forexample, archived biological samples (e.g., FFPE tissue sections) can betransferred from storage to a spatial array to perform spatial analysis.In some embodiments, a biological sample on a substrate can be coveredwith any of the prepolymer solutions described herein. In someembodiments, the prepolymer solution can be polymerized such that ahydrogel is formed on top of and/or around the biological sample.Hydrogel formation can occur in a manner sufficient to anchor (e.g.,embed) the biological sample to the hydrogel. After hydrogel formation,the biological sample is anchored to (e.g., embedded in) the hydrogelwherein separating the hydrogel from the substrate (e.g., glass slide)results in the biological sample separating from the substrate alongwith the hydrogel. The biological sample contained in the hydrogel canthen be contacted with a spatial array, and spatial analysis can beperformed on the biological sample.

Any variety of characteristics can determine the transfer conditionsrequired for a given biological sample. Non-limiting examples ofcharacteristics likely to impact transfer conditions include the sample(e.g., thickness, fixation, and cross-linking) and/or the analyte ofinterest (different conditions to preserve and/or transfer differentanalytes (e.g., DNA, RNA, and protein)).

In some embodiments, the hydrogel is removed after contacting thebiological sample with the spatial array. For example, methods describedherein can include an event-dependent (e.g., light or chemical)depolymerizing hydrogel, wherein upon application of the event (e.g.,external stimuli) the hydrogel depolymerizes. In one example, abiological sample can be anchored to a DTT-sensitive hydrogel, whereaddition of DTT can cause the hydrogel to depolymerize and release theanchored biological sample.

Hydrogels embedded within biological samples can be cleared using anysuitable method. For example, electrophoretic tissue clearing methodscan be used to remove biological macromolecules from thehydrogel-embedded sample. In some embodiments, a hydrogel-embeddedsample is stored in a medium before or after clearing of hydrogel (e.g.,a mounting medium, methylcellulose, or other semi-solid mediums).

In some embodiments, the hydrogel chemistry can be tuned to specificallybind (e.g., retain) particular species of analytes (e.g., RNA, DNA,protein, etc.). In some embodiments, a hydrogel includes a linker thatallows anchoring of the biological sample to the hydrogel. In someembodiments, a hydrogel includes linkers that allow anchoring ofbiological analytes to the hydrogel. In such cases, the linker can beadded to the hydrogel before, contemporaneously with, or after hydrogelformation. Non-limiting examples of linkers that anchor nucleic acids tothe hydrogel can include 6-((Acryloyl)amino) hexanoic acid (Acryloyl-XSE), Label-IT Amine and Label X (Chen et al., Nat. Methods 13:679-684,(2016)). Non-limiting examples of characteristics likely to impacttransfer conditions include the sample (e.g., thickness, fixation, andcross-linking) and/or the analyte of interest (different conditions topreserve and/or transfer different analytes (e.g., DNA, RNA, andprotein)).

Additional methods and aspects of hydrogel embedding of biologicalsamples are described for example in Chen et al., Science347(6221):543-548, 2015, the entire contents of which are incorporatedherein by reference.

(8) Biological Sample Transfer

In some embodiments, a biological sample immobilized on a substrate(e.g., a biological sample prepared using methanol fixation orformalin-fixation and paraffin-embedding (FFPE)) is transferred to aspatial array using a hydrogel. In some embodiments, a hydrogel isformed on top of a biological sample on a substrate (e.g., glass slide).For example, hydrogel formation can occur in a manner sufficient toanchor (e.g., embed) the biological sample to the hydrogel. Afterhydrogel formation, the biological sample is anchored to (e.g., embeddedin) the hydrogel wherein separating the hydrogel from the substrateresults in the biological sample separating from the substrate alongwith the hydrogel. The biological sample can then be contacted with aspatial array, thereby allowing spatial profiling of the biologicalsample. In some embodiments, the hydrogel is removed after contactingthe biological sample with the spatial array. For example, methodsdescribed herein can include an event-dependent (e.g., light orchemical) depolymerizing hydrogel, wherein upon application of the event(e.g., external stimuli) the hydrogel depolymerizes. In one example, abiological sample can be anchored to a DTT-sensitive hydrogel, whereaddition of DTT can cause the hydrogel to depolymerize and release theanchored biological sample. A hydrogel can be any appropriate hydrogelwhere upon formation of the hydrogel on the biological sample thebiological sample becomes anchored to or embedded in the hydrogel.Non-limiting examples of hydrogels are described herein or are known inthe art. In some embodiments, a hydrogel includes a linker that allowsanchoring of the biological sample to the hydrogel. In some embodiments,a hydrogel includes linkers that allow anchoring of biological analytesto the hydrogel. In such cases, the linker can be added to the hydrogelbefore, contemporaneously with, or after hydrogel formation.Non-limiting examples of linkers that anchor nucleic acids to thehydrogel can include 6-((Acryloyl)amino) hexanoic acid (Acryloyl-X SE)(available from ThermoFisher, Waltham, Mass.), Label-IT Amine (availablefrom MirusBio, Madison, Wis.) and Label X (Chen et al., Nat. Methods13:679-684, 2016). Any variety of characteristics can determine thetransfer conditions required for a given biological sample. Non-limitingexamples of characteristics likely to impact transfer conditions includethe sample (e.g., thickness, fixation, and cross-linking) and/or theanalyte of interest (different conditions to preserve and/or transferdifferent analytes (e.g., DNA, RNA, and protein)). In some embodiments,hydrogel formation can occur in a manner sufficient to anchor theanalytes (e.g., embed) in the biological sample to the hydrogel. In someembodiments, the hydrogel can be imploded (e.g., shrunk) with theanchored analytes (e.g., embedded in the hydrogel) present in thebiological sample. In some embodiments, the hydrogel can be expanded(e.g., isometric expansion) with the anchored analytes (e.g., embeddedin the hydrogel) present in the biological sample. In some embodiments,the hydrogel can be imploded (e.g., shrunk) and subsequently expandedwith anchored analytes (e.g., embedded in the hydrogel) present in thebiological sample.

(9) Isometric Expansion

In some embodiments, a biological sample embedded in a hydrogel can beisometrically expanded. Isometric expansion methods that can be usedinclude hydration, a preparative step in expansion microscopy, asdescribed in Chen et al., Science 347(6221):543-548, 2015; Asano et al.Current Protocols. 2018, 80:1, doi:10.1002/cpcb.56 and Gao et al. BMCBiology. 2017, 15:50, doi:10.1186/s12915-017-0393-3, Wassie, A. T., etal, Expansion microscopy: principles and uses in biological research,Nature Methods, 16(1): 33-41 (2018), each of which is incorporated byreference in its entirety.

In general, the steps used to perform isometric expansion of thebiological sample can depend on the characteristics of the sample (e.g.,thickness of tissue section, fixation, cross-linking), and/or theanalyte of interest (e.g., different conditions to anchor RNA, DNA, andprotein to a gel).

Isometric expansion can be performed by anchoring one or more componentsof a biological sample to a gel, followed by gel formation, proteolysis,and swelling. Isometric expansion of the biological sample can occurprior to immobilization of the biological sample on a substrate, orafter the biological sample is immobilized to a substrate. In someembodiments, the isometrically expanded biological sample can be removedfrom the substrate prior to contacting the expanded biological samplewith a spatially barcoded array (e.g., spatially barcoded capture probeson a substrate).

In some embodiments, proteins in the biological sample are anchored to aswellable gel such as a polyelectrolyte gel. An antibody can be directedto the protein before, after, or in conjunction with being anchored tothe swellable gel. DNA and/or RNA in a biological sample can also beanchored to the swellable gel via a suitable linker. Examples of suchlinkers include, but are not limited to, 6-((Acryloyl)amino) hexanoicacid (Acryloyl-X SE) (available from ThermoFisher, Waltham, Mass.),Label-IT Amine (available from MirusBio, Madison, Wis.) and Label X(described for example in Chen et al., Nat. Methods 13:679-684, 2016,the entire contents of which are incorporated herein by reference).

Isometric expansion of the sample can increase the spatial resolution ofthe subsequent analysis of the sample. For example, isometric expansionof the biological sample can result in increased resolution in spatialprofiling (e.g., single-cell profiling). The increased resolution inspatial profiling can be determined by comparison of an isometricallyexpanded sample with a sample that has not been isometrically expanded.

Isometric expansion can enable three-dimensional spatial resolution ofthe subsequent analysis of the sample. In some embodiments, isometricexpansion of the biological sample can occur in the presence of spatialprofiling reagents (e.g., analyte capture agents or capture probes). Forexample, the swellable gel can include analyte capture agents or captureprobes anchored to the swellable gel via a suitable linker. In someembodiments, spatial profiling reagents can be delivered to particularlocations in an isometrically expanded biological sample.

In some embodiments, a biological sample is isometrically expanded to avolume at least 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×,2.9×, 3×, 3.1×, 3.2×, 3.3×, 3.4×, 3.5×, 3.6×, 3.7×, 3.8×, 3.9×, 4×,4.1×, 4.2×, 4.3×, 4.4×, 4.5×, 4.6×, 4.7×, 4.8×, or 4.9× its non-expandedvolume. In some embodiments, the sample is isometrically expanded to atleast 2× and less than 20× of its non-expanded volume.

In some embodiments, a biological sample embedded in a hydrogel isisometrically expanded to a volume at least 2×, 2.1×, 2.2×, 2.3×, 2.4×,2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3×, 3.1×, 3.2×, 3.3×, 3.4×, 3.5×, 3.6×,3.7×, 3.8×, 3.9×, 4×, 4.1×, 4.2×, 4.3×, 4.4×, 4.5×, 4.6×, 4.7×, 4.8×, or4.9× its non-expanded volume. In some embodiments, the biological sampleembedded in a hydrogel is isometrically expanded to at least 2× and lessthan 20× of its non-expanded volume.

(10) Substrate Attachment

In some embodiments, the biological sample can be attached to asubstrate. Examples of substrates suitable for this purpose aredescribed in detail below. Attachment of the biological sample can beirreversible or reversible, depending upon the nature of the sample andsubsequent steps in the analytical method.

In certain embodiments, the sample can be attached to the substratereversibly by applying a suitable polymer coating to the substrate, andcontacting the sample to the polymer coating. The sample can then bedetached from the substrate using an organic solvent that at leastpartially dissolves the polymer coating. Hydrogels are examples ofpolymers that are suitable for this purpose.

More generally, in some embodiments, the substrate can be coated orfunctionalized with one or more substances to facilitate attachment ofthe sample to the substrate. Suitable substances that can be used tocoat or functionalize the substrate include, but are not limited to,lectins, poly-lysine, antibodies, and polysaccharides.

(11) Unaggregated Cells

In some embodiments, the biological sample corresponds to cells (e.g.,derived from a cell culture or a tissue sample). In a cell sample with aplurality of cells, individual cells can be naturally unaggregated. Forexample, cells can be derived from a suspension of cells and/ordisassociated or disaggregated cells from a tissue or tissue section.

Alternatively, the cells in the sample may be aggregated, and may bedisaggregated into individual cells using, for example, enzymatic ormechanical techniques. Examples of enzymes used in enzymaticdisaggregation include, but are not limited to, dispase, collagenase,trypsin, or combinations thereof. Mechanical disaggregation can beperformed, for example, using a tissue homogenizer.

In some embodiments of unaggregated cells or disaggregated cells, thecells are distributed onto the substrate such that at least one celloccupies a distinct spatial feature on the substrate. The cells can beimmobilized on the substrate (e.g., to prevent lateral diffusion of thecells). In some embodiments, a cell immobilization agent can be used toimmobilize a non-aggregated or disaggregated sample on aspatially-barcoded array prior to analyte capture. A “cellimmobilization agent” can refer to an antibody, attached to a substrate,which can bind to a cell surface marker. In some embodiments, thedistribution of the plurality of cells on the substrate follows Poissonstatistics.

In some embodiments, cells from a plurality of cells are immobilized ona substrate. In some embodiments, the cells are immobilized to preventlateral diffusion, for example, by adding a hydrogel and/or by theapplication of an electric field.

(12) Suspended and Adherent Cells

In some embodiments, the biological sample can be derived from a cellculture grown in vitro. Samples derived from a cell culture can includeone or more suspension cells which are anchorage-independent within thecell culture. Examples of such cells include, but are not limited to,cell lines derived from hematopoietic cells, and from the following celllines: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92,NCI-H322M, and MALME-3M.

Samples derived from a cell culture can include one or more adherentcells which grow on the surface of the vessel that contains the culturemedium.

(13) Tissue Permeabilization

In some embodiments, a biological sample can be permeabilized tofacilitate transfer of analytes out of the sample, and/or to facilitatetransfer of species (such as capture probes) into the sample. If asample is not permeabilized sufficiently, the amount of analyte capturedfrom the sample may be too low to enable adequate analysis. Conversely,if the tissue sample is too permeable, the relative spatial relationshipof the analytes within the tissue sample can be lost. Hence, a balancebetween permeabilizing the tissue sample enough to obtain good signalintensity while still maintaining the spatial resolution of the analytedistribution in the sample is desirable.

In general, a biological sample can be permeabilized by exposing thesample to one or more permeabilizing agents. Suitable agents for thispurpose include, but are not limited to, organic solvents (e.g.,acetone, ethanol, and methanol), cross-linking agents (e.g.,paraformaldehyde), detergents (e.g., saponin, Triton X-100™, Tween-20™,or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases(e.g., proteinase K). In some embodiments, the detergent is an anionicdetergent (e.g., SDS or N-lauroylsarcosine sodium salt solution). Insome embodiments, the biological sample can be permeabilized using anyof the methods described herein (e.g., using any of the detergentsdescribed herein, e.g., SDS and/or N-lauroylsarcosine sodium saltsolution) before or after enzymatic treatment (e.g., treatment with anyof the enzymes described herein, e.g., trypin, proteases (e.g., pepsinand/or proteinase K)).

In some embodiments, a biological sample can be permeabilized byexposing the sample to greater than about 1.0 w/v % (e.g., greater thanabout 2.0 w/v %, greater than about 3.0 w/v %, greater than about 4.0w/v %, greater than about 5.0 w/v %, greater than about 6.0 w/v %,greater than about 7.0 w/v %, greater than about 8.0 w/v %, greater thanabout 9.0 w/v %, greater than about 10.0 w/v %, greater than about 11.0w/v %, greater than about 12.0 w/v %, or greater than about 13.0 w/v %)sodium dodecyl sulfate (SDS) and/or N-lauroylsarcosine orN-lauroylsarcosine sodium salt. In some embodiments, a biological samplecan be permeabilized by exposing the sample (e.g., for about 5 minutesto about 1 hour, about 5 minutes to about 40 minutes, about 5 minutes toabout 30 minutes, about 5 minutes to about 20 minutes, or about 5minutes to about 10 minutes) to about 1.0 w/v % to about 14.0 w/v %(e.g., about 2.0 w/v % to about 14.0 w/v %, about 2.0 w/v % to about12.0 w/v %, about 2.0 w/v % to about 10.0 w/v %, about 4.0 w/v % toabout 14.0 w/v %, about 4.0 w/v % to about 12.0 w/v %, about 4.0 w/v %to about 10.0 w/v %, about 6.0 w/v % to about 14.0 w/v %, about 6.0 w/v% to about 12.0 w/v %, about 6.0 w/v % to about 10.0 w/v %, about 8.0w/v % to about 14.0 w/v %, about 8.0 w/v % to about 12.0 w/v %, about8.0 w/v % to about 10.0 w/v %, about 10.0% w/v % to about 14.0 w/v %,about 10.0 w/v % to about 12.0 w/v %, or about 12.0 w/v % to about 14.0w/v %) SDS and/or N-lauroylsarcosine salt solution and/or proteinase K(e.g., at a temperature of about 4% to about 35° C., about 4° C. toabout 25° C., about 4° C. to about 20° C., about 4° C. to about 10° C.,about 10° C. to about 25° C., about 10° C. to about 20° C., about 10° C.to about 15° C., about 35° C. to about 50° C., about 35° C. to about 45°C., about 35° C. to about 40° C., about 40° C. to about 50° C., about40° C. to about 45° C., or about 45° C. to about 50° C.).

In some embodiments, the biological sample can be incubated with apermeabilizing agent to facilitate permeabilization of the sample.Additional methods for sample permeabilization are described, forexample, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entirecontents of which are incorporated herein by reference.

Lysis Reagents

In some embodiments, the biological sample can be permeabilized byadding one or more lysis reagents to the sample. Examples of suitablelysis agents include, but are not limited to, bioactive reagents such aslysis enzymes that are used for lysis of different cell types, e.g.,gram positive or negative bacteria, plants, yeast, mammalian, such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other commercially available lysis enzymes.

Other lysis agents can additionally or alternatively be added to thebiological sample to facilitate permeabilization. For example,surfactant-based lysis solutions can be used to lyse sample cells. Lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). More generally, chemical lysis agentscan include, without limitation, organic solvents, chelating agents,detergents, surfactants, and chaotropic agents.

In some embodiments, the biological sample can be permeabilized bynon-chemical permeabilization methods. Non-chemical permeabilizationmethods are known in the art. For example, non-chemical permeabilizationmethods that can be used include, but are not limited to, physical lysistechniques such as electroporation, mechanical permeabilization methods(e.g., bead beating using a homogenizer and grinding balls tomechanically disrupt sample tissue structures), acousticpermeabilization (e.g., sonication), and thermal lysis techniques suchas heating to induce thermal permeabilization of the sample.

Proteases

In some embodiments, a medium, solution, or permeabilization solutionmay contain one or more proteases. In some embodiments, a biologicalsample treated with a protease capable of degrading histone proteins canresult in the generation of fragmented genomic DNA. The fragmentedgenomic DNA can be captured using the same capture domain (e.g., capturedomain having a poly(T) sequence) used to capture mRNA. In someembodiments, a biological sample is treated with a protease capable ofdegrading histone proteins and an RNA protectant prior to spatialprofiling in order to facilitate the capture of both genomic DNA andmRNA.

In some embodiments, a biological sample is permeabilized by exposingthe sample to a protease capable of degrading histone proteins. As usedherein, the term “histone protein” typically refers to a linker histoneprotein (e.g., H1) and/or a core histone protein (e.g., H2A, H2B, H3,and H4). In some embodiments, a protease degrades linker histoneproteins, core histone proteins, or linker histone proteins and corehistone proteins. Any suitable protease capable of degrading histoneproteins in a biological sample can be used. Non-limiting examples ofproteases capable of degrading histone proteins include proteasesinhibited by leupeptin and TLCK (Tosyl-L-lysyl-chloromethanehydrochloride), a protease encoded by the EUO gene from Chlamydiatrachomatis serovar A, granzyme A, a serine protease (e.g., trypsin ortrypsin-like protease, neutral serine protease, elastase, cathepsin G),an aspartyl protease (e.g., cathepsin D), a peptidase family C1 enzyme(e.g., cathepsin L), pepsin, proteinase K, a protease that is inhibitedby the diazomethane inhibitor Z-Phe-Phe-CHN(2) or the epoxide inhibitorE-64, a lysosomal protease, or an azurophilic enzyme (e.g., cathepsin G,elastase, proteinase 3, neutral serine protease). In some embodiments, aserine protease is a trypsin enzyme, trypsin-like enzyme or a functionalvariant or derivative thereof (e.g., P00761; C0HK48; Q8IYP2; Q8BW11;Q61E06; P35035; P00760; P06871; Q90627; P16049; P07477; P00762; P35031;P19799; P35036; Q29463; P06872; Q90628; P07478; P07146; P00763; P35032;P70059; P29786; P35037; Q90629; P35030; P08426; P35033; P35038; P12788;P29787; P35039; P35040; Q8NHM4; P35041; P35043; P35044; P54624; P04814;P35045; P32821; P54625; P35004; P35046; P32822; P35047; C0HKA5; C0HKA2;P54627; P35005; C0HKA6; C0HKA3; P52905; P83348; P00765; P35042; P81071;P35049; P51588; P35050; P35034; P35051; P24664; P35048; P00764; P00775;P54628; P42278; P54629; P42279; Q91041; P54630; P42280; C0HKA4) or acombination thereof. In some embodiments, a trypsin enzyme is P00761,P00760, Q29463, or a combination thereof. In some embodiments, aprotease capable of degrading one or more histone proteins comprises anamino acid sequence with at least 80% sequence identity to P00761,P00760, or Q29463. In some embodiments, a protease capable of degradingone or more histone proteins comprises an amino acid sequence with atleast 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto P00761, P00760, or Q29463. A protease may be considered a functionalvariant if it has at least 50% e.g., at least 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100% of the activity relative to the activity ofthe protease in condition optimum for the enzyme. In some embodiments,the enzymatic treatment with pepsin enzyme, or pepsin like enzyme, caninclude: P03954/PEPA1_MACFU; P28712/PEPA1_RABIT; P27677/PEPA2_MACFU;P27821/PEPA2_RABIT; P0DJD8/PEPA3_HUMAN; P27822/PEPA3_RABIT;P0DJD7/PEPA4_HUMAN; P27678/PEPA4_MACFU; P28713/PEPA4_RABIT;P0DJD9/PEPA5_HUMAN; Q9D106/PEPA5_MOUSE; P27823/PEPAF_RABIT;P00792/PEPA_BOVIN; Q9N2D4/PEPA_CALJA; Q9GMY6/PEPA_CANLF;P00793/PEPA_CHICK; P11489/PEPA_MACMU; P00791/PEPA_PIG;Q9GMY7/PEPA_RHIFE; Q9GMY8/PEPA_SORUN; P81497/PEPA_SUNMU;P13636/PEPA_URSTH and functional variants and derivatives thereof, or acombination thereof. In some embodiments, the pepsin enzyme can include:P00791/PEPA_PIG; P00792/PEPA_BOVIN, functional variants, derivatives, orcombinations thereof.

Additionally, the protease may be contained in a reaction mixture(solution), which also includes other components (e.g., buffer, salt,chelator (e.g., EDTA), and/or detergent (e.g., SDS, N-Lauroylsarcosinesodium salt solution)). The reaction mixture may be buffered, having apH of about 6.5-8.5, e.g., about 7.0-8.0. Additionally, the reactionmixture may be used at any suitable temperature, such as about 10-50°C., e.g., about 10-44° C., 11-43° C., 12-42° C., 13-41° C., 14-40° C.,15-39° C., 16-38° C., 17-37° C., e.g., about 10° C., 12° C., 15° C., 18°C., 20° C., 22° C., 25° C., 28° C., 30° C., 33° C., 35° C. or 37° C.,preferably about 35-45° C., e.g., about 37° C.

Other Reagents

In some embodiments, a permeabilization solution can contain additionalreagents or a biological sample may be treated with additional reagentsin order to optimize biological sample permeabilization. In someembodiments, an additional reagent is an RNA protectant. As used herein,the term “RNA protectant” typically refers to a reagent that protectsRNA from RNA nucleases (e.g., RNases). Any appropriate RNA protectantthat protects RNA from degradation can be used. A non-limiting exampleof a RNA protectant includes organic solvents (e.g., at least 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% v/v organic solvent), which include,without limitation, ethanol, methanol, propan-2-ol, acetone,trichloroacetic acid, propanol, polyethylene glycol, acetic acid, or acombination thereof. In some embodiments, a RNA protectant includesethanol, methanol and/or propan-2-ol, or a combination thereof. In someembodiments, a RNA protectant includes RNAlater ICE (ThermoFisherScientific). In some embodiments, the RNA protectant comprises at leastabout 60% ethanol. In some embodiments, the RNA protectant comprisesabout 60-95% ethanol, about 0-35% methanol and about 0-35% propan-2-ol,wherein the total amount of organic solvent in the medium is not morethan about 95%. In some embodiments, the RNA protectant comprises about60-95% ethanol, about 5-20% methanol and about 5-20% propan-2-ol,wherein the total amount of organic solvent in the medium is not morethan about 95%.

In some embodiments, the RNA protectant includes a salt. The salt mayinclude ammonium sulfate, ammonium bisulfate, ammonium chloride,ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron (II)sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II)sulfate, lithium chloride, lithium acetate, lithium sulfate, magnesiumsulfate, magnesium chloride, manganese sulfate, manganese chloride,potassium chloride, potassium sulfate, sodium chloride, sodium acetate,sodium sulfate, zinc chloride, zinc acetate and zinc sulfate. In someembodiments, the salt is a sulfate salt, for example, ammonium sulfate,ammonium bisulfate, cesium sulfate, cadmium sulfate, cesium iron (II)sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II)sulfate, lithium sulfate, magnesium sulfate, manganese sulfate,potassium sulfate, sodium sulfate, or zinc sulfate. In some embodiments,the salt is ammonium sulfate. The salt may be present at a concentrationof about 20 g/100 mL of medium or less, such as about 15 g/100 mL, 10g/100 mL, 9 g/100 mL, 8 g/100 mL, 7 g/100 mL, 6 g/100 mL, 5 g/100 mL orless, e.g., about 4 g/100 mL, 3 g/100 mL, 2 g/100 mL, or 1 g/100 mL.

Additionally, the RNA protectant may be contained in a medium thatfurther includes a chelator (e.g., EDTA), a buffer (e.g., sodiumcitrate, sodium acetate, potassium citrate, or potassium acetate,preferably sodium acetate), and/or buffered to a pH between about 4-8(e.g., about 5).

In some embodiments, the biological sample is treated with one or moreRNA protectants before, contemporaneously with, or afterpermeabilization. For example, a biological sample is treated with oneor more RNA protectants prior to treatment with one or morepermeabilization reagents (e.g., one or more proteases). In anotherexample, a biological sample is treated with a solution including one ormore RNA protectants and one or more permeabilization reagents (e.g.,one or more proteases). In yet another example, a biological sample istreated with one or more RNA protectants after the biological sample hasbeen treated with one or more permeabilization reagents (e.g., one ormore proteases). In some embodiments, a biological sample is treatedwith one or more RNA protectants prior to fixation.

In some embodiments, identifying the location of the captured analyte inthe biological sample includes a nucleic acid extension reaction. Insome embodiments where a capture probe captures a fragmented genomic DNAmolecule, a nucleic acid extension reaction includes DNA polymerase. Forexample, a nucleic acid extension reaction includes using a DNApolymerase to extend the capture probe that is hybridized to thecaptured analyte (e.g., fragmented genomic DNA) using the capturedanalyte (e.g., fragmented genomic DNA) as a template. The product of theextension reaction includes a spatially-barcoded analyte (e.g.,spatially-barcoded fragmented genomic DNA). The spatially-barcodedanalyte (e.g., spatially-barcoded fragmented genomic DNA) can be used toidentify the spatial location of the analyte in the biological sample.Any DNA polymerase that is capable of extending the capture probe usingthe captured analyte as a template can be used for the methods describedherein. Non-limiting examples of DNA polymerases include T7 DNApolymerase; Bsu DNA polymerase; and E. coli DNA Polymerase pol I.

Selective Permeabilization/Selective Lysis

In some embodiments, biological samples can be processed to selectivelyrelease an analyte from a subcellular region of a cell according toestablished methods. In some embodiments, a method provided herein caninclude detecting at least one biological analyte present in asubcellular region of a cell in a biological sample. As used herein, a“subcellular region” can refer to any subcellular region. For example, asubcellular region can refer to cytosol, a mitochondria, a nucleus, anucleolus, an endoplasmic reticulum, a lysosome, a vesicle, a Golgiapparatus, a plastid, a vacuole, a ribosome, cytoskeleton, orcombinations thereof. In some embodiments, the subcellular regioncomprises at least one of cytosol, a nucleus, a mitochondria, and amicrosome. In some embodiments, the subcellular region is cytosol. Insome embodiments, the subcellular region is a nucleus. In someembodiments, the subcellular region is a mitochondria. In someembodiments, the subcellular region is a microsome.

For example, a biological analyte can be selectively released from asubcellular region of a cell by selective permeabilization or selectivelysing. In some embodiments, “selective permeabilization” can refer to apermeabilization method that can permeabilize a membrane of asubcellular region while leaving a different subcellular regionsubstantially intact (e.g., biological analytes are not released fromsubcellular region due to the applied permeabilization method).Non-limiting examples of selective permeabilization methods includeusing electrophoresis and/or applying a permeabilization reagent. Insome embodiments, “selective lysing” can refer to a lysis method thatcan lyse a membrane of a subcellular region while leaving a differentsubcellular region substantially intact (e.g., biological analytes arenot released from subcellular region due to the applied lysis method).Several methods for selective permeabilization or lysis are known to oneof skill in the art including the methods described in Lu et al. LabChip. 2005 January; 5(1):23-9; Niklas et al. Anal Biochem. 2011 Sep. 15;416(2):218-27; Cox and Emili. Nat Protoc. 2006; 1(4):1872-8; Chiang etal. J Biochem. Biophys. Methods. 2000 Nov. 20; 46(1-2):53-68; andYamauchi and Herr et al. Microsyst. Nanoeng. 2017; 3. pii: 16079; eachof which is incorporated herein by reference in its entirety.

In some embodiments, “selective permeabilization” or “selective lysis”refer to the selective permeabilization or selective lysis of a specificcell type. For example, “selective permeabilization” or “selectivelysis” can refer to lysing one cell type while leaving a different celltype substantially intact (e.g., biological analytes are not releasedfrom the cell due to the applied permeabilization or lysis method). Acell that is a “different cell type” than another cell can refer to acell from a different taxonomic kingdom, a prokaryotic cell versus aeukaryotic cell, a cell from a different tissue type, etc. Many methodsare known to one of skill in the art for selectively permeabilizing orlysing different cell types. Non-limiting examples include applying apermeabilization reagent, electroporation, and/or sonication. See, e.g.,International Application No. WO 2012/168003; Han et al. MicrosystNanoeng. 2019 Jun. 17; 5:30; Gould et al. Oncotarget. 2018 Mar. 20;9(21): 15606-15615; Oren and Shai. Biochemistry. 1997 Feb. 18;36(7):1826-35; Algayer et al. Molecules. 2019 May 31; 24(11). pii:E2079; Hipp et al. Leukemia. 2017 October; 31(10):2278; InternationalApplication No. WO 2012/168003; and U.S. Pat. No. 7,785,869; all ofwhich are incorporated by reference herein in their entireties.

In some embodiments, applying a selective permeabilization or lysisreagent comprises contacting the biological sample with a hydrogelcomprising the permeabilization or lysis reagent.

In some embodiments, the biological sample is contacted with two or morearrays (e.g., flexible arrays, as described herein). For example, aftera subcellular region is permeabilized and a biological analyte from thesubcellular region is captured on a first array, the first array can beremoved, and a biological analyte from a different subcellular regioncan be captured on a second array.

(14) Selective Enrichment of RNA Species

In some embodiments, where RNA is the analyte, one or more RNA analytespecies of interest can be selectively enriched (e.g., Adiconis, et.al., Comparative analysis of RNA sequencing methods for degraded andlow-input samples, Nature, vol. 10, July 2013, 623-632, hereinincorporated by reference in its entirety). For example, one or morespecies of RNA can be selected by addition of one or moreoligonucleotides to the sample. In some embodiments, the additionaloligonucleotide is a sequence used for priming a reaction by apolymerase. For example, one or more primer sequences with sequencecomplementarity to one or more RNAs of interest can be used to amplifythe one or more RNAs of interest, thereby selectively enriching theseRNAs. In some embodiments, an oligonucleotide with sequencecomplementarity to the complementary strand of captured RNA (e.g., cDNA)can bind to the cDNA. For example, biotinylated oligonucleotides withsequence complementary to one or more cDNAs of interest binds to thecDNA and can be selected using biotinylation-streptavidin affinity usingany of a variety of methods known to the field (e.g., streptavidinbeads).

Alternatively, one or more species of RNA (e.g., ribosomal and/ormitochondrial RNA) can be down-selected (e.g., removed, depleted) usingany of a variety of methods. Non-limiting examples of a hybridizationand capture method of ribosomal RNA depletion include RiboMinus™,RiboCop™, and Ribo-Zero™. Another non-limiting RNA depletion methodinvolves hybridization of complementary DNA oligonucleotides to unwantedRNA followed by degradation of the RNA/DNA hybrids using RNase H.Non-limiting examples of a hybridization and degradation method includeNEBNext® rRNA depletion, NuGEN AnyDeplete, or RiboZero Plus. Anothernon-limiting ribosomal RNA depletion method includes ZapR™ digestion,for example SMARTer. In the SMARTer method, random nucleic acid adaptersare hybridized to RNA for first-strand synthesis and tailing by reversetranscriptase, followed by template switching and extension by reversetranscriptase. Additionally, first round PCR amplification addsfull-length Illumina sequencing adapters (e.g., Illumina indexes).Ribosomal RNA is cleaved by ZapR v2 and R probes v2. A second round ofPCR is performed, amplifying non-rRNA molecules (e.g., cDNA). Parts orsteps of these ribosomal depletion protocols/kits can be furthercombined with the methods described herein to optimize protocols for aspecific biological sample.

In depletion protocols, probes can be administered to a sample thatselectively hybridize to ribosomal RNA (rRNA), thereby reducing the pooland concentration of rRNA in the sample. Probes can be administered to abiological sample that selectively hybridize to mitochondria RNA(mtRNA), thereby reducing the pool and concentration of mtRNA in thesample. In some embodiments, probes complementary to mitochondrial RNAcan be added during cDNA synthesis, or probes complementary to bothribosomal and mitochondrial RNA can be added during cDNA synthesis.Subsequent application of capture probes to the sample can result inimproved capture of other types of RNA due to a reduction innon-specific RNA (e.g., down-selected RNA) present in the sample.Additionally and alternatively, duplex-specific nuclease (DSN) treatmentcan remove rRNA (see, e.g., Archer, et al, Selective and flexibledepletion of problematic sequences from RNA-seq libraries at the cDNAstage, BMC Genomics, 15 401, (2014), the entire contents of which areincorporated herein by reference). Furthermore, hydroxyapatitechromatography can remove abundant species (e.g., rRNA) (see, e.g.,Vandernoot, V. A., cDNA normalization by hydroxyapatite chromatographyto enrich transcriptome diversity in RNA-seq applications,Biotechniques, 53(6) 373-80, (2012), the entire contents of which areincorporated herein by reference).

(15) Other Reagents

Additional reagents can be added to a biological sample to performvarious functions prior to analysis of the biological sample. In someembodiments, nuclease inhibitors such as DNase and RNase inactivatingagents or protease inhibitors, and/or chelating agents such as EDTA, canbe added to the biological sample. In other embodiments nucleases, suchas DNase or RNAse, or proteases, such as pepsin or proteinase K, areadded to the sample. In some embodiments, additional reagents may bedissolved in a solution or applied as a medium to the sample. In someembodiments, additional reagents (e.g., pepsin) may be dissolved in HClprior to applying to the sample. For example, hematoxylin, from an H&Estain, can be optionally removed from the biological sample by washingin dilute HCl (0.001M to 0.1M) prior to further processing. In someembodiments, pepsin can be dissolved in dilute HCl (0.001M to 0.1M)prior to further processing. In some embodiments, biological samples canbe washed additional times (e.g., 2, 3, 4, 5, or more times) in diluteHCl prior to incubation with a protease (e.g., pepsin), but afterproteinase K treatment.

In some embodiments, the biological sample can be treated with one ormore enzymes. For example, one or more endonucleases to fragment DNA,DNA polymerase enzymes, and dNTPs used to amplify nucleic acids can beadded. Other enzymes that can also be added to the biological sampleinclude, but are not limited to, polymerase, transposase, ligase, andDNAse, and RNAse.

In some embodiments, reverse transcriptase enzymes can be added to thesample, including enzymes with terminal transferase activity, primers,and template switch oligonucleotides (TSOs). Template switching can beused to increase the length of a cDNA, e.g., by appending a predefinednucleic acid sequence to the cDNA. In some embodiments, the appendednucleic acid sequence comprises one or more ribonucleotides.

In some embodiments, additional reagents can be added to improve therecovery of one or more target molecules (e.g., cDNA molecules, mRNAtranscripts). For example, addition of carrier RNA to a RNA sampleworkflow process can increase the yield of extracted RNA/DNA hybridsfrom the biological sample. In some embodiments, carrier molecules areuseful when the concentration of input or target molecules is low ascompared to remaining molecules. Generally, single target moleculescannot form a precipitate, and addition of the carrier molecules canhelp in forming a precipitate. Some target molecule recovery protocolsuse carrier RNA to prevent small amounts of target nucleic acids presentin the sample from being irretrievably bound. In some embodiments,carrier RNA can be added immediately prior to a second strand synthesisstep. In some embodiments, carrier RNA can be added immediately prior toa second strand cDNA synthesis on oligonucleotides released from anarray. In some embodiments, carrier RNA can be added immediately priorto a post in vitro transcription clean-up step. In some embodiments,carrier RNA can be added prior to amplified RNA purification andquantification. In some embodiments, carrier

RNA can be added before RNA quantification. In some embodiments, carrierRNA can be added immediately prior to both a second strand cDNAsynthesis and a post in vitro transcription clean-up step.

(16) Pre-processing for Capture Probe Interaction

In some embodiments, analytes in a biological sample can bepre-processed prior to interaction with a capture probe. For example,prior to interaction with capture probes, polymerization reactionscatalyzed by a polymerase (e.g., DNA polymerase or reversetranscriptase) are performed in the biological sample. In someembodiments, a primer for the polymerization reaction includes afunctional group that enhances hybridization with the capture probe. Thecapture probes can include appropriate capture domains to capturebiological analytes of interest (e.g., poly(dT) sequence to capturepoly(A) mRNA).

In some embodiments, biological analytes are pre-processed for librarygeneration via next generation sequencing. For example, analytes can bepre-processed by addition of a modification (e.g., ligation of sequencesthat allow interaction with capture probes). In some embodiments,analytes (e.g., DNA or RNA) are fragmented using fragmentationtechniques (e.g., using transposases and/or fragmentation buffers).

Fragmentation can be followed by a modification of the analyte. Forexample, a modification can be the addition through ligation of anadapter sequence that allows hybridization with the capture probe. Insome embodiments, where the analyte of interest is RNA, poly(A) tailingis performed. Addition of a poly(A) tail to RNA that does not contain apoly(A) tail can facilitate hybridization with a capture probe thatincludes a capture domain with a functional amount of poly(dT) sequence.

In some embodiments, prior to interaction with capture probes, ligationreactions catalyzed by a ligase are performed in the biological sample.In some embodiments, ligation can be performed by chemical ligation. Insome embodiments, the ligation can be performed using click chemistry asfurther described below. In some embodiments, the capture domainincludes a DNA sequence that has complementarity to a RNA molecule,where the RNA molecule has complementarity to a second DNA sequence, andwhere the RNA-DNA sequence complementarity is used to ligate the secondDNA sequence to the DNA sequence in the capture domain. In theseembodiments, direct detection of RNA molecules is possible.

In some embodiments, prior to interaction with capture probes,target-specific reactions are performed in the biological sample.Examples of target specific reactions include, but are not limited to,ligation of target specific adaptors, probes and/or otheroligonucleotides, target specific amplification using primers specificto one or more analytes, and target-specific detection using in situhybridization, DNA microscopy, and/or antibody detection. In someembodiments, a capture probe includes capture domains targeted totarget-specific products (e.g., amplification or ligation).

II. General Spatial Array-Based Analytical Methodology

Provided herein are methods, apparatus, systems, and compositions forspatial array-based analysis of biological samples.

(a) Spatial Analysis Methods

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of each analyte within the biological sample. The spatiallocation of each analyte within the biological sample is determinedbased on the feature to which each analyte is bound on the array, andthe feature's relative spatial location within the array.

There are at least two general methods to associate a spatial barcodewith one or more neighboring cells, such that the spatial barcodeidentifies the one or more cells, and/or contents of the one or morecells, as associated with a particular spatial location. One generalmethod is to promote analytes out of a cell and towards thespatially-barcoded array. FIG. 1 depicts an exemplary embodiment of thisgeneral method. In FIG. 1, the spatially-barcoded array populated withcapture probes (as described further herein) is contacted with abiological sample 101, and biological sample is permeabilized, allowingthe analyte to migrate away from the sample and toward the array. Theanalyte interacts with a capture probe on the spatially-barcoded array102. Once the analyte hybridizes/is bound to the capture probe, thesample is optionally removed from the array and the capture probes areanalyzed in order to obtain spatially-resolved analyte information 103.

Another general method is to cleave the spatially-barcoded captureprobes from an array, and promote the spatially-barcoded capture probestowards and/or into or onto the biological sample. FIG. 2 depicts anexemplary embodiment of this general method, the spatially-barcodedarray populated with capture probes (as described further herein) can becontacted with a sample 201. The spatially-barcoded capture probes arecleaved and then interact with cells within the provided biologicalsample 202. The interaction can be a covalent or non-covalentcell-surface interaction. The interaction can be an intracellularinteraction facilitated by a delivery system or a cell penetrationpeptide. Once the spatially-barcoded capture probe is associated with aparticular cell, the sample can be optionally removed for analysis. Thesample can be optionally dissociated before analysis. Once the taggedcell is associated with the spatially-barcoded capture probe, thecapture probes can be analyzed to obtain spatially-resolved informationabout the tagged cell 203.

FIG. 3 shows an exemplary workflow that includes preparing a biologicalsample on a spatially-barcoded array 301. Sample preparation may includeplacing the sample on a slide, fixing the sample, and/or staining thebiological sample for imaging. The stained sample can be then imaged onthe array 302 using both brightfield (to image the sample hematoxylinand eosin stain) and/or fluorescence (to image features) modalities.Optionally, the sample can be destained prior to permeabilization. Insome embodiments, analytes are then released from the sample and captureprobes forming the spatially-barcoded array hybridize or bind thereleased analytes 303. The sample is then removed from the array 304 andthe capture probes cleaved from the array 305. The biological sample andarray are then optionally imaged a second time in one or both modalities305B while the analytes are reverse transcribed into cDNA, and anamplicon library is prepared 306 and sequenced 307. Images are thenspatially-overlaid in order to correlate spatially-identified biologicalsample information 308. When the sample and array are not imaged asecond time, 305B, a spot coordinate file is supplied instead. The spotcoordinate file replaces the second imaging step 305B. Further, ampliconlibrary preparation 306 can be performed with a unique PCR adapter andsequenced 307.

FIG. 4 shows another exemplary workflow that utilizes aspatially-barcoded array on a substrate, where spatially-barcodedcapture probes are clustered at areas called features. Thespatially-barcoded capture probes can include a cleavage domain, one ormore functional domains, a spatial barcode, a unique molecularidentifier, and a capture domain. The spatially-barcoded capture probescan also include a 5′ end modification for reversible attachment to thesubstrate. The spatially-barcoded array is contacted with a biologicalsample 401, and the sample is permeabilized through application ofpermeabilization reagents 402. Permeabilization reagents may beadministered by placing the array/sample assembly within a bulksolution. Alternatively, permeabilization reagents may be administeredto the sample via a diffusion-resistant medium and/or a physical barriersuch as a lid, wherein the sample is sandwiched between thediffusion-resistant medium and/or barrier and the array-containingsubstrate. The analytes are migrated toward the spatially-barcodedcapture array using any number of techniques disclosed herein. Forexample, analyte migration can occur using a diffusion-resistant mediumlid and passive migration. As another example, analyte migration can beactive migration, using an electrophoretic transfer system, for example.Once the analytes are in close proximity to the spatially-barcodedcapture probes, the capture probes can hybridize or otherwise bind atarget analyte 403. The biological sample can be optionally removed fromthe array 404.

The capture probes can be optionally cleaved from the array 405, and thecaptured analytes can be spatially-barcoded by performing a reversetranscriptase first strand cDNA reaction. A first strand cDNA reactioncan be optionally performed using template switching oligonucleotides.For example, a template switching oligonucleotide can hybridize to apoly(C) tail added to a 3′ end of the cDNA by a reverse transcriptaseenzyme in a template independent manner. The original mRNA template andtemplate switching oligonucleotide can then be denatured from the cDNAand the spatially-barcoded capture probe can then hybridize with thecDNA and a complement of the cDNA can be generated. The first strandcDNA can then be purified and collected for downstream amplificationsteps. The first strand cDNA can be amplified using PCR 406, where theforward and reverse primers flank the spatial barcode and analyteregions of interest, generating a library associated with a particularspatial barcode 407. In some embodiments, the library preparation can bequantitated and/or quality controlled to verify the success of thelibrary preparation steps 408. In some embodiments, the cDNA comprises asequencing by synthesis (SBS) primer sequence. The library amplicons aresequenced and analyzed to decode spatial information 407.

Using the methods, compositions, systems, kits, and devices describedherein, RNA transcripts present in biological samples (e.g., tissuesamples) can be used for spatial transcriptome analysis. In particular,in some cases, the barcoded oligonucleotides may be configured to prime,replicate, and consequently yield barcoded extension products from anRNA template, or derivatives thereof. For example, in some cases, thebarcoded oligonucleotides may include mRNA specific priming sequences,e.g., poly-T primer segments that allow priming and replication of mRNAin a reverse transcription reaction or other targeted priming sequences.Alternatively or additionally, random RNA priming may be carried outusing random N-mer primer segments of the barcoded oligonucleotides.Reverse transcriptases (RTs) can use an RNA template and a primercomplementary to the 3′ end of the RNA template to direct the synthesisof the first strand complementary DNA (cDNA). Many RTs can be used inthis reverse transcription reactions, including, for example, avianmyeloblastosis virus (AMV) reverse transcriptase, moloney murineleukemia virus (M-MuLV or MMLV), and other variants thereof. Somerecombinant M-MuLV reverse transcriptase, such as, for example,PROTOSCRIPT® II reverse transcriptase, can have reduced RNase H activityand increased thermostability when compared to its wild typecounterpart, and provide higher specificity, higher yield of cDNA andmore full-length cDNA products with up to 12 kilobase (kb) in length. Insome embodiments, the reverse transcriptase enzyme is a mutant reversetranscriptase enzyme such as, but not limited to, mutant MMLV reversetranscriptase. In another embodiment, the reverse transcriptase is amutant MMLV reverse transcriptase such as, but not limited to, one ormore variants described in US Patent Publication No. 20180312822 andU.S. Provisional Patent Application No. 62/946,885 filed on Dec. 11,2019, both of which are incorporated herein by reference in theirentireties.

FIG. 5 depicts an exemplary workflow where the biological sample isremoved from the spatially-barcoded array and the spatially-barcodedcapture probes are removed from the array for barcoded analyteamplification and library preparation. Another embodiment includesperforming first strand synthesis using template switchingoligonucleotides on the spatially-barcoded array without cleaving thecapture probes. In this embodiment, sample preparation 501 andpermeabilization 502 are performed as described elsewhere herein. Oncethe capture probes capture the analyte(s), first strand cDNA created bytemplate switching and reverse transcriptase 503 is then denatured andthe second strand is then extended 504. The second strand cDNA is thendenatured from the first strand cDNA, neutralized, and transferred to atube 505. cDNA quantification and amplification can be performed usingstandard techniques discussed herein. The cDNA can then be subjected tolibrary preparation 506 and indexing 507, including fragmentation,end-repair, and a-tailing, and indexing PCR steps. The librarypreparation can optionally be quality controlled to verify the successof the library preparation methods 508.

In a non-limiting example of the workflows described above, a biologicalsample (e.g., tissue section), can be fixed with methanol, stained withhematoxylin and eosin, and imaged. Optionally, the sample can bedestained prior to permeabilization. The images can be used to mapspatial gene expression patterns back to the biological sample. Apermeabilization enzyme can be used to permeabilize the biologicalsample directly on the slide. Analytes (e.g., polyadenylated mRNA)released from the overlying cells of the biological sample can becaptured by capture probes within a capture area on a substrate. Reversetranscription (RT) reagents can be added to permeabilized biologicalsamples. Incubation with the RT reagents can produce spatially-barcodedfull-length cDNA from the captured analytes (e.g., polyadenylated mRNA).Second strand reagents (e.g., second strand primers, enzymes) can beadded to the biological sample on the slide to initiate second strandsynthesis. The resulting cDNA can be denatured from the capture probetemplate and transferred (e.g., to a clean tube) for amplification,and/or library construction. The spatially-barcoded, full-length cDNAcan be amplified via PCR prior to library construction. The ampliconscan then be enzymatically fragmented and/or size-selected in order toprovide for desired amplicon size. In some embodiments, when utilizingan Illumina® library preparation methodology, P5 and P7 sequences can beadded to the amplifcons thereby allowing for capture of the librarypreparation on a sequencing flowcell (e.g., on Illumina sequencinginstruments). Additionally, i7 and i5 can index sequences be added assample indexes if multiple libraries are to be pooled and sequencedtogether. Further, Read 1 and Read 2 sequences can be added to thelibrary preparation for sequencing purposes. The aftorementionedsequences can be added to a library preparation sample, fore example,via End Repair, A-tailing, Adaptor Ligation, and/or PCR. The cDNAfragments can then be sequenced using, for example, paired-endsequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primersites.

In some embodiments, performing correlative analysis of data produced bythis workflow, and other workflows described herein, can yield over 95%correlation of genes expressed across two capture areas (e.g., 95% orgreater, 96% or greater, 97% or greater, 98% or greater, or 99% orgreater). When performing the described workflows using single cell RNAsequencing of nuclei, in some embodiments, correlative analysis of thedata can yield over 90% (e.g., over 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%) correlation of genes expressed across two captureareas.

In some embodiments, after cDNA is generated (e.g., by reversetranscription) the cDNA can be amplified directly on the substratesurface. Generating multiple copies of the cDNA (e.g., cDNA synthesizedfrom captured analytes) via amplification directly on the substratesurface can improve final sequencing library complexity. Thus, in someembodiments, cDNA can be amplified directly on the substrate surface byisothermal nucleic acid amplification. In some embodiments, isothermalnucleic acid amplification can amplify RNA or DNA.

In some embodiments, isothermal amplification can be faster than astandard PCR reaction. In some embodiments, isothermal amplification canbe linear amplification (e.g., asymmetrical with a single primer), orexponential amplification (e.g., with two primers). In some embodiments,isothermal nucleic acid amplification can be performed by atemplate-switching oligonucleotide primer. In some embodiments, thetemplate switching oligonucleotide adds a common sequence onto the 5′end of the RNA being reverse transcribed. For example, after a captureprobe interacts with an analyte (e.g., mRNA) and reverse transcriptionis performed such that additional nucleotides are added to the end ofthe cDNA creating a 3′ overhang as described herein. In someembodiments, a template switching oligonucleotide hybridizes tountemplated poly(C) nucleotides added by a reverse transcriptase tocontinue replication to the 5′ end of the template switchingoligonucleotide, thereby generating full-length cDNA ready for furtheramplification. In some embodiments, the template switchingoligonucleotide adds a common 5′ sequence to full-length cDNA that isused for cDNA amplification (e.g., a reverse complement of the templateswitching oligonucleotide).

In some embodiments, once a full-length cDNA molecule is generated, thetemplate switching oligonucleotide can serve as a primer in a cDNAamplification reaction (e.g., with a DNA polymerase). In someembodiments, double stranded cDNA (e.g., first strand cDNA and secondstrand reverse complement cDNA) can be amplified via isothermalamplification with either a helicase or recombinase, followed by astrand displacing DNA polymerase. The strand displacing DNA polymerasecan generate a displaced second strand resulting in an amplifiedproduct.

In any of isothermal amplification methods described herein, barcodeexchange (e.g., spatial barcode) can occur after the first amplificationcycle where there are unused capture probes on the substrate surface. Insome embodiments, the free 3′OH end of the unused capture probes can beblocked by any suitable 3′OH blocking method. In some embodiments, the3′OH can be blocked by hairpin ligation.

Isothermal nucleic acid amplification can be used in addition to, or asan alternative to standard PCR reactions (e.g., a PCR reaction thatrequires heating to about 95° C. to denature double stranded DNA).Isothermal nucleic acid amplification generally does not require the useof a thermocycler, however in some embodiments, isothermal amplificationcan be performed in a thermocycler. In some embodiments, isothermalamplification can be performed from about 35° C. to about 75° C. In someembodiments, isothermal amplification can be performed from about 40°C., about 45° C., about 50° C., about 55° C., about 60° C., about 65°C., or about 70° C. or anywhere in between depending on the polymeraseand auxiliary enzymes used.

Isothermal nucleic acid amplification techniques are known in the art,and can be used alone or in combination with any of the spatial methodsdescribed herein. For example, non-limiting examples of suitableisothermal nucleic acid amplification techniques include transcriptionmediated amplification, nucleic acid sequence-based amplification,signal mediated amplification of RNA technology, strand displacementamplification, rolling circle amplification, loop-mediated isothermalamplification of DNA (LAMP), isothermal multiple displacementamplification, recombinase polymerase amplification, helicase-dependentamplification, single primer isothermal amplification, and circularhelicase-dependent amplification (See, e.g., Gill and Ghaemi, Nucleicacid isothermal amplification technologies: a review, Nucleosides,Nucleotides, & Nucleic Acids, 27(3), 224-43, doi:10.1080/15257770701845204 (2008), which is incorporated herein byreference in its entirety).

In some embodiments, the isothermal nucleic acid amplification ishelicase-dependent nucleic acid amplification. Helicase-dependentisothermal nucleic acid amplification is described in Vincent, et. al.,Helicase-dependent isothermal DNA amplification, EMBO Rep., 795-800(2004) and U.S. Pat. No. 7,282,328, which are both incorporated hereinby reference in their entireties. Further, helicase-dependent nucleicacid amplification on a substrate (e.g., on-chip) is described inAndresen, et. al., Helicase-dependent amplification: use in OnChipamplification and potential for point-of-care diagnostics, Expert RevMol Diagn., 9, 645-650, doi: 10.1586/erm.09.46 (2009), which isincorporated herein by reference in its entirety. In some embodiments,the isothermal nucleic acid amplification is recombinase polymerasenucleic acid amplification. Recombinase polymerase nucleic acidamplification is described in Piepenburg, et al., DNA Detection UsingRecombinant Proteins, PLoS Biol., 4, 7 e204 (2006) and Li, et. al.,Review: a comprehensive summary of a decade development of therecombinase polymerase amplification, Analyst, 144, 31-67, doi:10.1039/C8AN01621F (2019), both of which are incorporated herein byreference in their entireties.

Generally, isothermal amplification techniques use standard PCR reagents(e.g., buffer, dNTPs etc.) known in the art. Some isothermalamplification techniques can require additional reagents. For example,helicase dependent nucleic acid amplification uses a single-strandbinding protein and an accessory protein. In another example,recombinase polymerase nucleic acid amplification uses recombinase(e.g., T4 UvsX), recombinase loading factor (e.g., TF UvsY),single-strand binding protein (e.g., T4 gp32), crowding agent (e.g.,PEG-35K), and ATP.

After isothermal nucleic acid amplification of the full-length cDNAdescribed by any of the methods herein, the isothermally amplified cDNAs(e.g., single-stranded or double-stranded) can be recovered from thesubstrate, and optionally followed by amplification with typical cDNAPCR in microcentrifuge tubes. The sample can then be used with any ofthe spatial methods described herein.

(i) Immunohistochemistry and Immunofluorescence

In some embodiments, immunofluorescence or immunohistochemistryprotocols (direct and indirect staining techniques) can be performed asa part of, or in addition to, the exemplary spatial workflows presentedherein. For example, tissue sections can be fixed according to methodsdescribed herein. The biological sample can be transferred to an array(e.g., capture probe array), wherein analytes (e.g., proteins) areprobed using immunofluorescence protocols. For example, the sample canbe rehydrated, blocked, and permeabilized (3×SSC, 2% BSA, 0.1% Triton X,1 U/μl RNAse inhibitor for 10 min at 4° C.) before being stained withfluorescent primary antibodies (1:100 in 3×SSC, 2% BSA, 0.1% Triton X, 1U/μl RNAse inhibitor for 30 min at 4° C.). The biological sample can bewashed, coverslipped (in glycerol+1 U/μl RNAse inhibitor), imaged (e.g.,using a confocal microscope or other apparatus capable of fluorescentdetection), washed, and processed according to analyte capture orspatial workflows described herein.

As used herein, an “antigen retrieval buffer” can improve antibodycapture in IF/IHC protocols. An exemplary protocol for antigen retrievalcan be preheating the antigen retrieval buffer (e.g., to 95° C.),immersing the biological sample in the heated antigen retrieval bufferfor a predetermined time, and then removing the biological sample fromthe antigen retrieval buffer and washing the biological sample.

In some embodiments, optimizing permeabilization can be useful foridentifying intracellular analytes. Permeabilization optimization caninclude selection of permeabilization agents, concentration ofpermeabilization agents, and permeabilization duration. Tissuepermeabilization is discussed elsewhere herein.

In some embodiments, blocking an array and/or a biological sample inpreparation of labeling the biological sample decreases unspecificbinding of the antibodies to the array and/or biological sample(decreases background). Some embodiments provide for blockingbuffers/blocking solutions that can be applied before and/or duringapplication of the label, wherein the blocking buffer can include ablocking agent, and optionally a surfactant and/or a salt solution. Insome embodiments, a blocking agent can be bovine serum albumin (BSA),serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk),casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), orpolyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidaseblocking reagent, levamisole, Carnoy's solution, glycine, lysine, sodiumborohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blockingagent, and/or acetic acid. The blocking buffer/blocking solution can beapplied to the array and/or biological sample prior to and/or duringlabeling (e.g., application of fluorophore-conjugated antibodies) to thebiological sample.

In some embodiments, additional steps or optimizations can be includedin performing IF/IHC protocols in conjunction with spatial arrays.Additional steps or optimizations can be included in performingspatially-tagged analyte capture agent workflows discussed herein.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., an analyte presentin a biological sample, such as a tissue section) that include: (a)providing a biological sample on a substrate; (b) staining thebiological sample on the substrate, imaging the stained biologicalsample, and selecting the biological sample or subsection of thebiological sample (e.g., region of interest) to subject to analysis; (c)providing an array comprising one or more pluralities of capture probeson a substrate; (d) contacting the biological sample with the array,thereby allowing a capture probe of the one or more pluralities ofcapture probes to capture the analyte of interest; and (e) analyzing thecaptured analyte, thereby spatially detecting the analyte of interest.Any variety of staining and imaging techniques as described herein orknown in the art can be used in accordance with methods describedherein. In some embodiments, the staining includes optical labels asdescribed herein, including, but not limited to, fluorescent,radioactive, chemiluminescent, calorimetric, or colorimetric detectablelabels. In some embodiments, the staining includes a fluorescentantibody directed to a target analyte (e.g., cell surface orintracellular proteins) in the biological sample. In some embodiments,the staining includes an immunohistochemistry stain directed to a targetanalyte (e.g., cell surface or intracellular proteins) in the biologicalsample. In some embodiments, the staining includes a chemical stain suchas hematoxylin and eosin (H&E) or periodic acid-schiff (PAS). In someembodiments, significant time (e.g., days, months, or years) can elapsebetween staining and/or imaging the biological sample and performinganalysis. In some embodiments, reagents for performing analysis areadded to the biological sample before, contemporaneously with, or afterthe array is contacted to the biological sample. In some embodiments,step (d) includes placing the array onto the biological sample. In someembodiments, the array is a flexible array where the plurality ofspatially-barcoded features (e.g., a substrate with capture probes, abead with capture probes) are attached to a flexible substrate. In someembodiments, measures are taken to slow down a reaction (e.g., coolingthe temperature of the biological sample or using enzymes thatpreferentially perform their primary function at lower or highertemperature as compared to their optimal functional temperature) beforethe array is contacted with the biological sample. In some embodiments,step (e) is performed without bringing the biological sample out ofcontact with the array. In some embodiments, step (e) is performed afterthe biological sample is no longer in contact with the array. In someembodiments, the biological sample is tagged with an analyte captureagent before, contemporaneously with, or after staining and/or imagingof the biological sample. In such cases, significant time (e.g., days,months, or years) can elapse between staining and/or imaging andperforming analysis. In some embodiments, the array is adapted tofacilitate biological analyte migration from the stained and/or imagedbiological sample onto the array (e.g., using any of the materials ormethods described herein). In some embodiments, a biological sample ispermeabilized before being contacted with an array. In some embodiments,the rate of permeabilization is slowed prior to contacting a biologicalsample with an array (e.g., to limit diffusion of analytes away fromtheir original locations in the biological sample). In some embodiments,modulating the rate of permeabilization (e.g., modulating the activityof a permeabilization reagent) can occur by modulating a condition thatthe biological sample is exposed to (e.g., modulating temperature, pH,and/or light). In some embodiments, modulating the rate ofpermeabilization includes use of external stimuli (e.g., smallmolecules, enzymes, and/or activating reagents) to modulate the rate ofpermeabilization. For example, a permeabilization reagent can beprovided to a biological sample prior to contact with an array, whichpermeabilization reagent is inactive until a condition (e.g.,temperature, pH, and/or light) is changed or an external stimulus (e.g.,a small molecule, an enzyme, and/or an activating reagent) is provided.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample such as a tissue section) that include: (a) providinga biological sample on a substrate; (b) staining the biological sampleon the substrate, imaging the stained biological sample, and selectingthe biological sample or subsection of the biological sample (e.g., aregion of interest) to subject to spatial transcriptomic analysis; (c)providing an array comprising one or more pluralities of capture probeson a substrate; (d) contacting the biological sample with the array,thereby allowing a capture probe of the one or more pluralities ofcapture probes to capture the biological analyte of interest; and (e)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte of interest.

(b) Capture Probes

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe is a conjugate (e.g., an oligonucleotide-antibody conjugate). Insome embodiments, the capture probe includes a barcode (e.g., a spatialbarcode and/or a unique molecular identifier (UMI)) and a capturedomain.

FIG. 6 is a schematic diagram showing an example of a capture probe, asdescribed herein. As shown, the capture probe 602 is optionally coupledto a feature 601 by a cleavage domain 603, such as a disulfide linker.The capture probe can include functional sequences that are useful forsubsequent processing, such as functional sequence 604, which caninclude a sequencer specific flow cell attachment sequence, e.g., a P5or P7 sequence, as well as functional sequence 606, which can includesequencing primer sequences, e.g., a R1 primer binding site, a R2 primerbinding site. In some embodiments, sequence 604 is a P7 sequence andsequence 606 is a R2 primer binding site. A spatial barcode 605 can beincluded within the capture probe for use in barcoding the targetanalyte. The functional sequences can generally be selected forcompatibility with any of a variety of different sequencing systems,e.g., Ion Torrent Proton or PGM, Illumina sequencing instruments,PacBio, Oxford Nanopore, etc., and the requirements thereof. In someembodiments, functional sequences can be selected for compatibility withnon-commercialized sequencing systems. Examples of such sequencingsystems and techniques, for which suitable functional sequences can beused, include (but are not limited to) Ion Torrent Proton or PGMsequencing, Illumina sequencing, PacBio SMRT sequencing, and OxfordNanopore sequencing. Further, in some embodiments, functional sequencescan be selected for compatibility with other sequencing systems,including non-commercialized sequencing systems.

In some embodiments, the spatial barcode 605, functional sequences 604(e.g., flow cell attachment sequence) and 606 (e.g., sequencing primersequences) can be common to all of the probes attached to a givenfeature. The spatial barcode can also include a capture domain 607 tofacilitate capture of a target analyte.

(i) Capture Domain

As discussed above, each capture probe includes at least one capturedomain. The “capture domain” can be an oligonucleotide, a polypeptide, asmall molecule, or any combination thereof, that binds specifically to adesired analyte. In some embodiments, a capture domain can be used tocapture or detect a desired analyte.

In some embodiments, the capture domain is a functional nucleic acidsequence configured to interact with one or more analytes, such as oneor more different types of nucleic acids (e.g., RNA molecules and DNAmolecules). In some embodiments, the functional nucleic acid sequencecan include an N-mer sequence (e.g., a random N-mer sequence), whichN-mer sequences are configured to interact with a plurality of DNAmolecules. In some embodiments, the functional sequence can include apoly(T) sequence, which poly(T) sequences are configured to interactwith messenger RNA (mRNA) molecules via the poly(A) tail of an mRNAtranscript. In some embodiments, the functional nucleic acid sequence isthe binding target of a protein (e.g., a transcription factor, a DNAbinding protein, or a RNA binding protein), where the analyte ofinterest is a protein.

Capture probes can include ribonucleotides and/or deoxyribonucleotidesas well as synthetic nucleotide residues that are capable ofparticipating in Watson-Crick type or analogous base pair interactions.In some embodiments, the capture domain is capable of priming a reversetranscription reaction to generate cDNA that is complementary to thecaptured RNA molecules. In some embodiments, the capture domain of thecapture probe can prime a DNA extension (polymerase) reaction togenerate DNA that is complementary to the captured DNA molecules. Insome embodiments, the capture domain can template a ligation reactionbetween the captured DNA molecules and a surface probe that is directlyor indirectly immobilized on the substrate. In some embodiments, thecapture domain can be ligated to one strand of the captured DNAmolecules. For example, SplintR ligase along with RNA or DNA sequences(e.g., degenerate RNA) can be used to ligate a single-stranded DNA orRNA to the capture domain. In some embodiments, ligases withRNA-templated ligase activity, e.g., SplintR ligase, T4 RNA ligase 2 orKOD ligase, can be used to ligate a single-stranded DNA or RNA to thecapture domain. In some embodiments, a capture domain includes a splintoligonucleotide. In some embodiments, a capture domain captures a splintoligonucleotide.

In some embodiments, the capture domain is located at the 3′ end of thecapture probe and includes a free 3′ end that can be extended, e.g., bytemplate dependent polymerization, to form an extended capture probe asdescribed herein. In some embodiments, the capture domain includes anucleotide sequence that is capable of hybridizing to nucleic acid,e.g., RNA or other analyte, present in the cells of the biologicalsample contacted with the array. In some embodiments, the capture domaincan be selected or designed to bind selectively or specifically to atarget nucleic acid. For example, the capture domain can be selected ordesigned to capture mRNA by way of hybridization to the mRNA poly(A)tail. Thus, in some embodiments, the capture domain includes a poly(T)DNA oligonucleotide, e.g., a series of consecutive deoxythymidineresidues linked by phosphodiester bonds, which is capable of hybridizingto the poly(A) tail of mRNA. In some embodiments, the capture domain caninclude nucleotides that are functionally or structurally analogous to apoly(T) tail. For example, a poly(U) oligonucleotide or anoligonucleotide included of deoxythymidine analogues. In someembodiments, the capture domain includes at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capturedomain includes at least 25, 30, or 35 nucleotides.

In some embodiments, a capture probe includes a capture domain having asequence that is capable of binding to mRNA and/or genomic DNA. Forexample, the capture probe can include a capture domain that includes anucleic acid sequence (e.g., a poly(T) sequence) capable of binding to apoly(A) tail of an mRNA and/or to a poly(A) homopolymeric sequencepresent in genomic DNA. In some embodiments, a homopolymeric sequence isadded to an mRNA molecule or a genomic DNA molecule using a terminaltransferase enzyme in order to produce an analyte that has a poly(A) orpoly(T) sequence. For example, a poly(A) sequence can be added to ananalyte (e.g., a fragment of genomic DNA) thereby making the analytecapable of capture by a poly(T) capture domain.

In some embodiments, random sequences, e.g., random hexamers or similarsequences, can be used to form all or a part of the capture domain. Forexample, random sequences can be used in conjunction with poly(T) (orpoly(T) analogue) sequences. Thus, where a capture domain includes apoly(T) (or a “poly(T)-like”) oligonucleotide, it can also include arandom oligonucleotide sequence (e.g., “poly(T)-random sequence” probe).This can, for example, be located 5′ or 3′ of the poly(T) sequence,e.g., at the 3′ end of the capture domain. The poly(T)-random sequenceprobe can facilitate the capture of the mRNA poly(A) tail. In someembodiments, the capture domain can be an entirely random sequence. Insome embodiments, degenerate capture domains can be used.

In some embodiments, a pool of two or more capture probes form amixture, where the capture domain of one or more capture probes includesa poly(T) sequence and the capture domain of one or more capture probesincludes random sequences. In some embodiments, a pool of two or morecapture probes form a mixture where the capture domain of one or morecapture probes includes poly(T)-like sequence and the capture domain ofone or more capture probes includes random sequences. In someembodiments, a pool of two or more capture probes form a mixture wherethe capture domain of one or more capture probes includes apoly(T)-random sequences and the capture domain of one or more captureprobes includes random sequences. In some embodiments, probes withdegenerate capture domains can be added to any of the precedingcombinations listed herein. In some embodiments, probes with degeneratecapture domains can be substituted for one of the probes in each of thepairs described herein.

The capture domain can be based on a particular gene sequence orparticular motif sequence or common/conserved sequence, that it isdesigned to capture (i.e., a sequence-specific capture domain). Thus, insome embodiments, the capture domain is capable of binding selectivelyto a desired sub-type or subset of nucleic acid, for example aparticular type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA,snoRNA, SmY RNA, scaRNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA,cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA,7SK, eRNA, ncRNA or other types of RNA. In a non-limiting example, thecapture domain can be capable of binding selectively to a desired subsetof ribonucleic acids, for example, microbiome RNA, such as 16S rRNA.

In some embodiments, a capture domain includes an “anchor” or “anchoringsequence”, which is a sequence of nucleotides that is designed to ensurethat the capture domain hybridizes to the intended analyte. In someembodiments, an anchor sequence includes a sequence of nucleotides,including a 1-mer, 2-mer, 3-mer or longer sequence. In some embodiments,the short sequence is random. For example, a capture domain including apoly(T) sequence can be designed to capture an mRNA. In suchembodiments, an anchoring sequence can include a random 3-mer (e.g.,GGG) that helps ensure that the poly(T) capture domain hybridizes to anmRNA. In some embodiments, an anchoring sequence can be VN, N, or NN.Alternatively, the sequence can be designed using a specific sequence ofnucleotides. In some embodiments, the anchor sequence is at the 3′ endof the capture domain. In some embodiments, the anchor sequence is atthe 5′ end of the capture domain.

In some embodiments, capture domains of capture probes are blocked priorto contacting the biological sample with the array, and blocking probesare used when the nucleic acid in the biological sample is modifiedprior to its capture on the array. In some embodiments, the blockingprobe is used to block or modify the free 3′ end of the capture domain.In some embodiments, blocking probes can be hybridized to the captureprobes to mask the free 3′ end of the capture domain, e.g., hairpinprobes, partially double stranded probes, or complementary sequences. Insome embodiments, the free 3′ end of the capture domain can be blockedby chemical modification, e.g., addition of an azidomethyl group as achemically reversible capping moiety such that the capture probes do notinclude a free 3′ end. Blocking or modifying the capture probes,particularly at the free 3′ end of the capture domain, prior tocontacting the biological sample with the array, prevents modificationof the capture probes, e.g., prevents the addition of a poly(A) tail tothe free 3′ end of the capture probes.

Non-limiting examples of 3′ modifications include dideoxy C-3′ (3′-ddC),3′ inverted dT, 3′ C3 spacer, 3′ Amino, and 3′ phosphorylation. In someembodiments, the nucleic acid in the biological sample can be modifiedsuch that it can be captured by the capture domain. For example, anadaptor sequence (including a binding domain capable of binding to thecapture domain of the capture probe) can be added to the end of thenucleic acid, e.g., fragmented genomic DNA. In some embodiments, this isachieved by ligation of the adaptor sequence or extension of the nucleicacid. In some embodiments, an enzyme is used to incorporate additionalnucleotides at the end of the nucleic acid sequence, e.g., a poly(A)tail. In some embodiments, the capture probes can be reversibly maskedor modified such that the capture domain of the capture probe does notinclude a free 3′ end. In some embodiments, the 3′ end is removed,modified, or made inaccessible so that the capture domain is notsusceptible to the process used to modify the nucleic acid of thebiological sample, e.g., ligation or extension.

In some embodiments, the capture domain of the capture probe is modifiedto allow the removal of any modifications of the capture probe thatoccur during modification of the nucleic acid molecules of thebiological sample. In some embodiments, the capture probes can includean additional sequence downstream of the capture domain, e.g., 3′ to thecapture domain, namely a blocking domain.

In some embodiments, the capture domain of the capture probe can be anon-nucleic acid domain. Examples of suitable capture domains that arenot exclusively nucleic-acid based include, but are not limited to,proteins, peptides, aptamers, antigens, antibodies, and molecularanalogs that mimic the functionality of any of the capture domainsdescribed herein.

(ii) Cleavage Domain

Each capture probe can optionally include at least one cleavage domain.The cleavage domain represents the portion of the probe that is used toreversibly attach the probe to an array feature, as will be describedfurther herein. Further, one or more segments or regions of the captureprobe can optionally be released from the array feature by cleavage ofthe cleavage domain. As an example, spatial barcodes and/or universalmolecular identifiers (UMIs) can be released by cleavage of the cleavagedomain.

FIG. 7 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to analytes within the sample. The capture probe 701 contains acleavage domain 702, a cell penetrating peptide 703, a reporter molecule704, and a disulfide bond (—S—S—). 705 represents all other parts of acapture probe, for example a spatial barcode and a capture domain.

In some embodiments, the cleavage domain linking the capture probe to afeature is a bond capable of cleavage by an enzyme. An enzyme can beadded to cleave the cleavage domain, resulting in release of the captureprobe from the feature. As another example, heating can also result indegradation of the cleavage domain and release of the attached captureprobe from the array feature. In some embodiments, laser radiation isused to heat and degrade cleavage domains of capture probes at specificlocations. In some embodiments, the cleavage domain is a photo-sensitivechemical bond (e.g., a chemical bond that dissociates when exposed tolight such as ultraviolet light). In some embodiments, the cleavagedomain can be an ultrasonic cleavage domain. For example, ultrasoniccleavage can depend on nucleotide sequence, length, pH, ionic strength,temperature, and the ultrasonic frequency (e.g., 22 kHz, 44 kHz)(Grokhovsky, S. L., Specificity of DNA cleavage by ultrasound, MolecularBiology, 40(2), 276-283 (2006)).

Oligonucleotides with photo-sensitive chemical bonds (e.g.,photo-cleavable linkers) have various advantages. They can be cleavedefficiently and rapidly (e.g., in nanoseconds and milliseconds). In somecases, photo-masks can be used such that only specific regions of thearray are exposed to cleavable stimuli (e.g., exposure to UV light,exposure to light, exposure to heat induced by laser). When aphoto-cleavable linker is used, the cleavable reaction is triggered bylight, and can be highly selective to the linker and consequentlybiorthogonal. Typically, wavelength absorption for the photocleavablelinker is located in the near-UV range of the spectrum. In someembodiments, λ_(max) of the photocleavable linker is from about 300 nmto about 400 nm, or from about 310 nm to about 365 nm. In someembodiments, λmax of the photocleavable linker is about 300 nm, about312 nm, about 325 nm, about 330 nm, about 340 nm, about 345 nm, about355 nm, about 365 nm, or about 400 nm.

Non-limiting examples of a photo-sensitive chemical bond that can beused in a cleavage domain include those described in Leriche et al.Bioorg Med Chem. 2012 Jan. 15; 20(2):571-82 and U.S. Publication No.2017/0275669, both of which are incorporated by reference herein intheir entireties.

Other examples of photo-sensitive chemical bonds that can be used in acleavage domain include halogenated nucleosides such asbromodeoxyuridine (BrdU). BrdU is an analog of thymidine that can bereadily incorporated into oligonucleotides (e.g., in the cleavage domainof a capture probe), and is sensitive to UVB light (280-320 nm range).Upon exposure to UVB light, a photo-cleavage reaction occurs (e.g., at anucleoside immediately 5′ to the site of BrdU incorporation (Doddridgeet al. Chem. Comm., 1998, 18:1997-1998 and Cook et al. Chemistry andBiology. 1999, 6:451-459)) that results in release of the capture probefrom the feature.

Other examples of cleavage domains include labile chemical bonds suchas, but not limited to, ester linkages (e.g., cleavable with an acid, abase, or hydroxylamine), a vicinal diol linkage (e.g., cleavable viasodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), asulfone linkage (e.g., cleavable via a base), a silyl ether linkage(e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable viaan amylase), a peptide linkage (e.g., cleavable via a protease), anabasic or apurinic/apyrimidinic (AP) site (e.g., cleavable with analkali or an AP endonuclease), or a phosphodiester linkage (e.g.,cleavable via a nuclease (e.g., DNAase)).

In some embodiments, the cleavage domain includes a sequence that isrecognized by one or more enzymes capable of cleaving a nucleic acidmolecule, e.g., capable of breaking the phosphodiester linkage betweentwo or more nucleotides. A bond can be cleavable via other nucleic acidmolecule targeting enzymes, such as restriction enzymes (e.g.,restriction endonucleases). For example, the cleavage domain can includea restriction endonuclease (restriction enzyme) recognition sequence.Restriction enzymes cut double-stranded or single stranded DNA atspecific recognition nucleotide sequences known as restriction sites. Insome embodiments, a rare-cutting restriction enzyme, e.g., enzymes witha long recognition site (at least 8 base pairs in length), is used toreduce the possibility of cleaving elsewhere in the capture probe.

In some embodiments, the cleavage domain includes a poly(U) sequencewhich can be cleaved by a mixture of Uracil DNA glycosylase (UDG) andthe DNA glycosylase-lyase Endonuclease VIII, commercially known as theUSER™ enzyme. Releasable capture probes can be available for reactiononce released. Thus, for example, an activatable capture probe can beactivated by releasing the capture probes from a feature.

In some embodiments, where the capture probe is attached indirectly to asubstrate, e.g., via a surface probe, the cleavage domain includes oneor more mismatch nucleotides, so that the complementary parts of thesurface probe and the capture probe are not 100% complementary (forexample, the number of mismatched base pairs can be one, two, or threebase pairs). Such a mismatch is recognized, e.g., by the MutY and T7endonuclease I enzymes, which results in cleavage of the nucleic acidmolecule at the position of the mismatch. As described herein a “surfaceprobe” can be any moiety present on the surface of the substrate capableof attaching to an agent (e.g., a capture probe). In some embodiments,the surface probe is an oligonucleotide. In some embodiments, thesurface probe is part of the capture probe.

In some embodiments, where the capture probe is attached (e.g.,immobilized) to a feature indirectly, e.g., via a surface probe, thecleavage domain includes a nickase recognition site or sequence.Nickases are endonucleases which cleave only a single strand of a DNAduplex. Thus, the cleavage domain can include a nickase recognition siteclose to the 5′ end of the surface probe (and/or the 5′ end of thecapture probe) such that cleavage of the surface probe or capture probedestabilizes the duplex between the surface probe and capture probethereby releasing the capture probe) from the feature.

Nickase enzymes can also be used in some embodiments where the captureprobe is attached (e.g., immobilized) to the feature directly. Forexample, the substrate can be contacted with a nucleic acid moleculethat hybridizes to the cleavage domain of the capture probe to provideor reconstitute a nickase recognition site, e.g., a cleavage helperprobe. Thus, contact with a nickase enzyme will result in cleavage ofthe cleavage domain thereby releasing the capture probe from thefeature. Such cleavage helper probes can also be used to provide orreconstitute cleavage recognition sites for other cleavage enzymes,e.g., restriction enzymes.

Some nickases introduce single-stranded nicks only at particular siteson a DNA molecule, by binding to and recognizing a particular nucleotiderecognition sequence. A number of naturally-occurring nickases have beendiscovered, of which at present the sequence recognition properties havebeen determined for at least four. Nickases are described in U.S. Pat.No. 6,867,028, which is incorporated herein by reference in itsentirety. In general, any suitable nickase can be used to bind to acomplementary nickase recognition site of a cleavage domain. Followinguse, the nickase enzyme can be removed from the assay or inactivatedfollowing release of the capture probes to prevent unwanted cleavage ofthe capture probes.

Examples of suitable capture domains that are not exclusivelynucleic-acid based include, but are not limited to, proteins, peptides,aptamers, antigens, antibodies, and molecular analogs that mimic thefunctionality of any of the capture domains described herein.

In some embodiments, a cleavage domain is absent from the capture probe.Examples of substrates with attached capture probes lacking a cleavagedomain are described for example in Macosko et al., (2015) Cell 161,1202-1214, the entire contents of which are incorporated herein byreference.

In some embodiments, the region of the capture probe corresponding tothe cleavage domain can be used for some other function. For example, anadditional region for nucleic acid extension or amplification can beincluded where the cleavage domain would normally be positioned. In suchembodiments, the region can supplement the functional domain or evenexist as an additional functional domain. In some embodiments, thecleavage domain is present but its use is optional.

(iii) Functional Domain

Each capture probe can optionally include at least one functionaldomain. Each functional domain typically includes a functionalnucleotide sequence for a downstream analytical step in the overallanalysis procedure.

In some embodiments, the capture probe can include a functional domainfor attachment to a sequencing flow cell, such as, for example, a P5sequence for Illumina® sequencing. In some embodiments, the captureprobe or derivative thereof can include another functional domain, suchas, for example, a P7 sequence for attachment to a sequencing flow cellfor Illumina® sequencing. The functional domains can be selected forcompatibility with a variety of different sequencing systems, e.g., 454Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and therequirements thereof.

In some embodiments, the functional domain includes a primer. The primercan include an R1 primer sequence for Illumina® sequencing, and in someembodiments, an R2 primer sequence for Illumina® sequencing. Examples ofsuch capture probes and uses thereof are described in U.S. PatentPublication Nos. 2014/0378345 and 2015/0376609, the entire contents ofeach of which are incorporated herein by reference.

(iv) Spatial Barcode

As discussed above, the capture probe can include one or more spatialbarcodes (e.g., two or more, three or more, four or more, five or more)spatial barcodes. A “spatial barcode” is a contiguous nucleic acidsegment or two or more non-contiguous nucleic acid segments thatfunction as a label or identifier that conveys or is capable ofconveying spatial information. In some embodiments, a capture probeincludes a spatial barcode that possesses a spatial aspect, where thebarcode is associated with a particular location within an array or aparticular location on a substrate.

A spatial barcode can be part of an analyte, or independent from ananalyte (e.g., part of the capture probe). A spatial barcode can be atag attached to an analyte (e.g., a nucleic acid molecule) or acombination of a tag in addition to an endogenous characteristic of theanalyte (e.g., size of the analyte or end sequence(s)). A spatialbarcode can be unique. In some embodiments where the spatial barcode isunique, the spatial barcode functions both as a spatial barcode and as aunique molecular identifier (UMI), associated with one particularcapture probe.

Spatial barcodes can have a variety of different formats. For example,spatial barcodes can include polynucleotide spatial barcodes; randomnucleic acid and/or amino acid sequences; and synthetic nucleic acidand/or amino acid sequences. In some embodiments, a spatial barcode isattached to an analyte in a reversible or irreversible manner. In someembodiments, a spatial barcode is added to, for example, a fragment of aDNA or RNA sample before, during, and/or after sequencing of the sample.In some embodiments, a spatial barcode allows for identification and/orquantification of individual sequencing-reads. In some embodiments, aspatial barcode is a used as a fluorescent barcode for whichfluorescently labeled oligonucleotide probes hybridize to the spatialbarcode.

In some embodiments, the spatial barcode is a nucleic acid sequence thatdoes not substantially hybridize to analyte nucleic acid molecules in abiological sample. In some embodiments, the spatial barcode has lessthan 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than40% sequence identity) to the nucleic acid sequences across asubstantial part (e.g., 80% or more) of the nucleic acid molecules inthe biological sample.

The spatial barcode sequences can include from about 6 to about 20 ormore nucleotides within the sequence of the capture probes. In someembodiments, the length of a spatial barcode sequence can be about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.In some embodiments, the length of a spatial barcode sequence can be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some embodiments, the length of a spatialbarcode sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 nucleotides or shorter.

These nucleotides can be completely contiguous, e.g., in a singlestretch of adjacent nucleotides, or they can be separated into two ormore separate subsequences that are separated by 1 or more nucleotides.Separated spatial barcode subsequences can be from about 4 to about 16nucleotides in length. In some embodiments, the spatial barcodesubsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or longer. In some embodiments, the spatial barcodesubsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some embodiments, the spatial barcodesubsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or shorter.

For multiple capture probes that are attached to a common array feature,the one or more spatial barcode sequences of the multiple capture probescan include sequences that are the same for all capture probes coupledto the feature, and/or sequences that are different across all captureprobes coupled to the feature.

FIG. 8 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature. In FIG. 8, the feature 801 can be coupled tospatially-barcoded capture probes, wherein the spatially-barcoded probesof a particular feature can possess the same spatial barcode, but havedifferent capture domains designed to associate the spatial barcode ofthe feature with more than one target analyte. For example, a featuremay be coupled to four different types of spatially-barcoded captureprobes, each type of spatially-barcoded capture probe possessing thespatial barcode 802. One type of capture probe associated with thefeature includes the spatial barcode 802 in combination with a poly(T)capture domain 803, designed to capture mRNA target analytes. A secondtype of capture probe associated with the feature includes the spatialbarcode 802 in combination with a random N-mer capture domain 804 forgDNA analysis. A third type of capture probe associated with the featureincludes the spatial barcode 802 in combination with a capture domaincomplementary to the analyte capture agent of interest 805. A fourthtype of capture probe associated with the feature includes the spatialbarcode 802 in combination with a capture probe that can specificallybind a nucleic acid molecule 806 that can function in a CRISPR assay(e.g., CRISPR/Cas9). While only four different capture probe-barcodedconstructs are shown in FIG. 8, capture-probe barcoded constructs can betailored for analyses of any given analyte associated with a nucleicacid and capable of binding with such a construct. For example, theschemes shown in FIG. 8 can also be used for concurrent analysis ofother analytes disclosed herein, including, but not limited to: (a)mRNA, a lineage tracing construct, cell surface or intracellularproteins and metabolites, and gDNA; (b) mRNA, accessible chromatin(e.g., ATAC-seq, DNase-seq, and/or MNase-seq) cell surface orintracellular proteins and metabolites, and a perturbation agent (e.g.,a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein); (c) mRNA, cell surface orintracellular proteins and/or metabolites, a barcoded labelling agent(e.g., the MHC multimers described herein), and a V(D)J sequence of animmune cell receptor (e.g., T-cell receptor). In some embodiments, aperturbation agent can be a small molecule, an antibody, a drug, anaptamer, a miRNA, a physical environmental (e.g., temperature change),or any other known perturbation agents.

Capture probes attached to a single array feature can include identical(or common) spatial barcode sequences, different spatial barcodesequences, or a combination of both. Capture probes attached to afeature can include multiple sets of capture probes. Capture probes of agiven set can include identical spatial barcode sequences. The identicalspatial barcode sequences can be different from spatial barcodesequences of capture probes of another set.

The plurality of capture probes can include spatial barcode sequences(e.g., nucleic acid barcode sequences) that are associated with specificlocations on a spatial array. For example, a first plurality of captureprobes can be associated with a first region, based on a spatial barcodesequence common to the capture probes within the first region, and asecond plurality of capture probes can be associated with a secondregion, based on a spatial barcode sequence common to the capture probeswithin the second region. The second region may or may not be associatedwith the first region. Additional pluralities of capture probes can beassociated with spatial barcode sequences common to the capture probeswithin other regions. In some embodiments, the spatial barcode sequencescan be the same across a plurality of capture probe molecules.

In some embodiments, multiple different spatial barcodes areincorporated into a single arrayed capture probe. For example, a mixedbut known set of spatial barcode sequences can provide a strongeraddress or attribution of the spatial barcodes to a given spot orlocation, by providing duplicate or independent confirmation of theidentity of the location. In some embodiments, the multiple spatialbarcodes represent increasing specificity of the location of theparticular array point.

(v) Unique Molecular Identifier

The capture probe can include one or more (e.g., two or more, three ormore, four or more, five or more) Unique Molecular Identifiers (UMIs). Aunique molecular identifier is a contiguous nucleic acid segment or twoor more non-contiguous nucleic acid segments that function as a label oridentifier for a particular analyte, or for a capture probe that binds aparticular analyte (e.g., via the capture domain).

A UMI can be unique. A UMI can include one or more specificpolynucleotides sequences, one or more random nucleic acid and/or aminoacid sequences, and/or one or more synthetic nucleic acid and/or aminoacid sequences, or combinations thereof.

In some embodiments, the UMI is a nucleic acid sequence that does notsubstantially hybridize to analyte nucleic acid molecules in abiological sample. In some embodiments, the UMI has less than 80%sequence identity (e.g., less than 70%, 60%, 50%, or less than 40%sequence identity) to the nucleic acid sequences across a substantialpart (e.g., 80% or more) of the nucleic acid molecules in the biologicalsample.

The UMI can include from about 6 to about 20 or more nucleotides withinthe sequence of the capture probes. In some embodiments, the length of aUMI sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 nucleotides or longer. In some embodiments, the length of aUMI sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, thelength of a UMI sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

These nucleotides can be completely contiguous, i.e., in a singlestretch of adjacent nucleotides, or they can be separated into two ormore separate subsequences that are separated by 1 or more nucleotides.Separated UMI subsequences can be from about 4 to about 16 nucleotidesin length. In some embodiments, the UMI subsequence can be about 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In someembodiments, the UMI subsequence can be at least about 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments,the UMI subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 nucleotides or shorter.

In some embodiments, a UMI is attached to an analyte in a reversible orirreversible manner. In some embodiments, a UMI is added to, forexample, a fragment of a DNA or RNA sample before, during, and/or aftersequencing of the analyte. In some embodiments, a UMI allows foridentification and/or quantification of individual sequencing-reads. Insome embodiments, a UMI is a used as a fluorescent barcode for whichfluorescently labeled oligonucleotide probes hybridize to the UMI.

(vi) Other Aspects of Capture Probes

For capture probes that are attached to an array feature, an individualarray feature can include one or more capture probes. In someembodiments, an individual array feature includes hundreds or thousandsof capture probes. In some embodiments, the capture probes areassociated with a particular individual feature, where the individualfeature contains a capture probe including a spatial barcode unique to adefined region or location on the array.

In some embodiments, a particular feature can contain capture probesincluding more than one spatial barcode (e.g., one capture probe at aparticular feature can include a spatial barcode that is different thanthe spatial barcode included in another capture probe at the sameparticular feature, while both capture probes include a second, commonspatial barcode), where each spatial barcode corresponds to a particulardefined region or location on the array. For example, multiple spatialbarcode sequences associated with one particular feature on an array canprovide a stronger address or attribution to a given location byproviding duplicate or independent confirmation of the location. In someembodiments, the multiple spatial barcodes represent increasingspecificity of the location of the particular array point. In anon-limiting example, a particular array point can be coded with twodifferent spatial barcodes, where each spatial barcode identifies aparticular defined region within the array, and an array pointpossessing both spatial barcodes identifies the sub-region where twodefined regions overlap, e.g., such as the overlapping portion of a Venndiagram.

In another non-limiting example, a particular array point can be codedwith three different spatial barcodes, where the first spatial barcodeidentifies a first region within the array, the second spatial barcodeidentifies a second region, where the second region is a subregionentirely within the first region, and the third spatial barcodeidentifies a third region, where the third region is a subregionentirely within the first and second subregions.

In some embodiments, capture probes attached to array features arereleased from the array features for sequencing. Alternatively, in someembodiments, capture probes remain attached to the array features, andthe probes are sequenced while remaining attached to the array features(e.g., via in situ sequencing). Further aspects of the sequencing ofcapture probes are described in subsequent sections of this disclosure.

In some embodiments, an array feature can include different types ofcapture probes attached to the feature. For example, the array featurecan include a first type of capture probe with a capture domain designedto bind to one type of analyte, and a second type of capture probe witha capture domain designed to bind to a second type of analyte. Ingeneral, array features can include one or more (e.g., two or more,three or more, four or more, five or more, six or more, eight or more,ten or more, 12 or more, 15 or more, 20 or more, 30 or more, 50 or more)different types of capture probes attached to a single array feature.

In some embodiments, the capture probe is nucleic acid. In someembodiments, the capture probe is attached to the array feature via its5′ end. In some embodiments, the capture probe includes from the 5′ to3′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI) andone or more capture domains. In some embodiments, the capture probeincludes from the 5′ to 3′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probeincludes from the 5′ to 3′ end: a cleavage domain, a functional domain,one or more barcodes (e.g., a spatial barcode and/or a UMI), and acapture domain. In some embodiments, the capture probe includes from the5′ to 3′ end: a cleavage domain, a functional domain, one or morebarcodes (e.g., a spatial barcode and/or a UMI), a second functionaldomain, and a capture domain. In some embodiments, the capture probeincludes from the 5′ to 3′ end: a cleavage domain, a functional domain,a spatial barcode, a UMI, and a capture domain. In some embodiments, thecapture probe does not include a spatial barcode. In some embodiments,the capture probe does not include a UMI. In some embodiments, thecapture probe includes a sequence for initiating a sequencing reaction.

In some embodiments, the capture probe is immobilized on a feature viaits 3′ end. In some embodiments, the capture probe includes from the 3′to 5′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI)and one or more capture domains. In some embodiments, the capture probeincludes from the 3′ to 5′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probeincludes from the 3′ to 5′ end: a cleavage domain, a functional domain,one or more barcodes (e.g., a spatial barcode and/or a UMI), and acapture domain. In some embodiments, the capture probe includes from the3′ to 5′ end: a cleavage domain, a functional domain, a spatial barcode,a UMI, and a capture domain.

In some embodiments, a capture probe includes an in situ synthesizedoligonucleotide. The in situ synthesized oligonucleotide can be attachedto a substrate, or to a feature on a substrate. In some embodiments, thein situ synthesized oligonucleotide includes one or more constantsequences, one or more of which serves as a priming sequence (e.g., aprimer for amplifying target nucleic acids). The in situ synthesizedoligonucleotide can, for example, include a constant sequence at the 3′end that is attached to a substrate, or attached to a feature on asubstrate. Additionally or alternatively, the in situ synthesizedoligonucleotide can include a constant sequence at the free 5′ end. Insome embodiments, the one or more constant sequences can be a cleavablesequence. In some embodiments, the in situ synthesized oligonucleotideincludes a barcode sequence, e.g., a variable barcode sequence. Thebarcode can be any of the barcodes described herein. The length of thebarcode can be approximately 8 to 16 nucleotides (e.g., 8, 9, 10, 11,12, 13, 14, 15, or 16 nucleotides). The length of the in situsynthesized oligonucleotide can be less than 100 nucleotides (e.g., lessthan 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25 or 20 nucleotides). Insome instances, the length of the in situ synthesized oligonucleotide isabout 20 to about 40 nucleotides. Exemplary in situ synthesizedoligonucleotides are produced by Affymetrix. In some embodiments, the insitu synthesized oligonucleotide is attached to a feature of an array.

Additional oligonucleotides can be ligated to an in situ synthesizedoligonucleotide to generate a capture probe. For example, a primercomplementary to a portion of the in situ synthesized oligonucleotide(e.g., a constant sequence in the oligonucleotide) can be used tohybridize an additional oligonucleotide and extend (using the in situsynthesized oligonucleotide as a template e.g., a primer extensionreaction) to form a double stranded oligonucleotide and to furthercreate a 3′ overhang. In some embodiments, the 3′ overhang can becreated by template-independent ligases (e.g., terminal deoxynucleotidyltransferase (TdT) or poly(A) polymerase). An additional oligonucleotidecomprising one or more capture domains can be ligated to the 3′ overhangusing a suitable enzyme (e.g., a ligase) and a splint oligonucleotide,to generate a capture probe. Thus, in some embodiments, a capture probeis a product of two or more oligonucleotide sequences, (e.g., the insitu synthesized oligonucleotide and the additional oligonucleotide)that are ligated together. In some embodiments, one of theoligonucleotide sequences is an in situ synthesized oligonucleotide.

In some embodiments, the capture probe can be prepared using a splintoligonucleotide (e.g., any of the splint oligonucleotides describedherein). Two or more oligonucleotides can be ligated together using asplint oligonucleotide and any variety of ligases known in the art ordescribed herein (e.g., SplintR ligase).

One of the oligonucleotides can include, for example, a constantsequence (e.g., a sequence complementary to a portion of a splintoligonucleotide), a degenerate sequence, and/or a capture domain (e.g.,as described herein). One of the oligonucleotides can also include asequence compatible for ligating or hybridizing to an analyte ofinterest in the biological sample. An analyte of interest (e.g., anmRNA) can also be used as a splint oligonucleotide to ligate furtheroligonucleotides onto the capture probe. In some embodiments, thecapture probe is generated by having an enzyme add polynucleotides atthe end of an oligonucleotide sequence. The capture probe can include adegenerate sequence, which can function as a unique molecularidentifier.

A degenerate sequence, which is a sequence in which some positions of anucleotide sequence contain a number of possible bases. A degeneratesequence can be a degenerate nucleotide sequence including about or atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, or 50 nucleotides. In some embodiments, a nucleotidesequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or moredegenerate positions within the nucleotide sequence. In someembodiments, the degenerate sequence is used as a UMI.

In some embodiments, a capture probe includes a restriction endonucleaserecognition sequence or a sequence of nucleotides cleavable by specificenzyme activities. For example, uracil sequences can be enzymaticallycleaved from a nucleotide sequence using uracil DNA glycosylase (UDG) orUracil Specific Excision Reagent (USER). As another example, othermodified bases (e.g., modified by methylation) can be recognized andcleaved by specific endonucleases. The capture probes can be subjectedto an enzymatic cleavage, which removes the blocking domain and any ofthe additional nucleotides that are added to the 3′ end of the captureprobe during the modification process. Removal of the blocking domainreveals and/or restores the free 3′ end of the capture domain of thecapture probe. In some embodiments, additional nucleotides can beremoved to reveal and/or restore the 3′ end of the capture domain of thecapture probe.

In some embodiments, a blocking domain can be incorporated into thecapture probe when it is synthesized, or after its synthesis. Theterminal nucleotide of the capture domain is a reversible terminatornucleotide (e.g., 3′-O-blocked reversible terminator and 3′-unblockedreversible terminator), and can be included in the capture probe duringor after probe synthesis.

(vii) Extended Capture Probes

An “extended capture probe” is a capture probe with an enlarged nucleicacid sequence. For example, where the capture probe includes nucleicacid, an “extended 3′ end” indicates that further nucleotides were addedto the most 3′ nucleotide of the capture probe to extend the length ofthe capture probe, for example, by standard polymerization reactionsutilized to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase orreverse transcriptase).

In some embodiments, extending the capture probe includes generatingcDNA from the captured (hybridized) RNA. This process involves synthesisof a complementary strand of the hybridized nucleic acid, e.g.,generating cDNA based on the captured RNA template (the RNA hybridizedto the capture domain of the capture probe). Thus, in an initial step ofextending the capture probe, e.g., the cDNA generation, the captured(hybridized) nucleic acid, e.g., RNA, acts as a template for theextension, e.g., reverse transcription, step.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, the capture domain of the capture probe includes aprimer for producing the complementary strand of the nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA, e.g., cDNA, molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if the nucleicacid, e.g., RNA, was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from Lucigen,Middleton, Wis.). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended capture probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9° N™ DNAligase, New England Biolabs), Ampligase™ (available from Lucigen,Middleton, Wis.), and SplintR (available from New England Biolabs,Ipswich, Mass.). In some embodiments, a polynucleotide tail, e.g., apoly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g., sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to an array feature specific for the affinitygroup. In some embodiments, the substrate can include an antibody orantibody fragment. In some embodiments, the array feature includesavidin or streptavidin and the affinity group includes biotin. In someembodiments, the array feature includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the array featureincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the array feature, insofaras copies of the extended probes are not attached to the array feature.

In some embodiments, the extended capture probe or complement oramplicon thereof is released from an array feature. The step ofreleasing the extended capture probe or complement or amplicon thereoffrom an array feature can be achieved in a number of ways. In someembodiments, an extended capture probe or a complement thereof isreleased from the feature by nucleic acid cleavage and/or bydenaturation (e.g., by heating to denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the array feature by physical means.For example, methods for inducing physical release include denaturingdouble stranded nucleic acid molecules. Another method for releasing theextended capture probes is to use a solution that interferes with thehydrogen bonds of the double stranded molecules. In some embodiments,the extended capture probe is released by applying heated water such aswater or buffer of at least 85° C., e.g., at least 90, 91, 92, 93, 94,95, 96, 97, 98, or 99° C. In some embodiments, a solution includingsalts, surfactants, etc. that can further destabilize the interactionbetween the nucleic acid molecules is added to release the extendedcapture probe from the array feature. In some embodiments, a formamidesolution can be used to destabilize the interaction between nucleic acidmolecules to release the extended capture probe from the array feature.

(viii) Amplification of Capture Probes

In some embodiments, methods are provided herein for amplifying acapture probe affixed to a spatial array, where amplification of thecapture probe increases the number of capture domains and spatialbarcodes on the spatial array. In some embodiments where a capture probeis amplified, the amplification is performed by rolling circleamplification. In some embodiments, the capture probe to be amplifiedincludes sequences (e.g., docking sequences, functional sequences,and/or primer sequences) that enable rolling circle amplification. Inone example, the capture probe can include a functional sequence that iscapable of binding to a primer used for amplification. In anotherexample, the capture probe can include one or more docking sequences(e.g., a first docking sequence and a second docking sequence) that canhybridize to one or more oligonucleotides (e.g., a padlock probe(s))used for rolling circle amplification. In some embodiments, additionalprobes are affixed to the substrate, where the additional probes includesequences (e.g., a docking sequence(s), a functional sequence(s), and/ora primer sequence(s)) that enable rolling circle amplification. In someembodiments, the spatial array is contacted with an oligonucleotide(e.g., a padlock probe). As used herein, a “padlock probe” refers to anoligonucleotide that has, at its 5′ and 3′ ends, sequences that arecomplementary to adjacent or nearby target sequences (e.g., dockingsequences) on a capture probe. Upon hybridization to the targetsequences (e.g., docking sequences), the two ends of the padlock probeare either brought into contact or an end is extended until the two endsare brought into contact, allowing circularization of the padlock probeby ligation (e.g., ligation using any of the methods described herein).In some embodiments, after circularization of the oligonucleotide,rolling circle amplification can be used to amplify the ligationproduct, which includes at least a capture domain and a spatial barcodefrom the capture probe. In some embodiments, amplification of thecapture probe using a padlock oligonucleotide and rolling circleamplification increases the number of capture domains and the number ofspatial barcodes on the spatial array.

In some embodiments, a method of increasing capture efficiency of aspatial array includes amplifying all or part of a capture probe affixedto a substrate. For example, amplification of all or part of the captureprobes affixed to the substrate can increase the capture efficiency ofthe spatial array by increasing the number of capture domains andspatial barcodes. In some embodiments, a method of determining alocation of an analyte in a biological sample includes using a spatialarray having increased capture efficiency (e.g., a spatial array where acapture probe has been amplified as described herein). For example, thecapture efficiency of a spatial array can be increased by amplificationof all or part of the capture probe prior to contact with a biologicalsample. The amplification results in an increased number of capturedomains that enable capture of more analytes as compared to a spatialarray where the capture probe was not amplified prior to contacting thebiological sample. In some embodiments, a method of producing a spatialarray that has increased capture efficiency includes amplifying all orpart of a capture probe. In some embodiments where a spatial arrayhaving increased capture efficiency is produced by amplifying all orpart of a capture probe, the amplification increases the number ofcapture domains and the number of spatial barcodes on the spatial array.In some embodiments, a method of determining the location of a captureprobe (i.e., a capture probe on a feature) on a spatial array includesamplifying all or part of a capture probe. For example, amplification ofthe capture probe affixed to the substrate can increase the number ofspatial barcodes used for direct decoding (e.g., direct decoding usingany of the methods described herein including, without limitation, insitu sequencing) of the location of the capture probe.

(ix) Analyte Capture Agents

This disclosure also provides methods and materials for using analytecapture agents for spatial profiling of biological analytes (e.g., mRNA,genomic DNA, accessible chromatin, and cell surface or intracellularproteins and/or metabolites). As used herein, an “analyte capture agent”(also referred to previously at times as a “cell labelling” agent”)refers to an agent that interacts with an analyte (e.g., an analyte in asample) and with a capture probe (e.g., a capture probe attached to asubstrate) to identify the analyte. In some embodiments, the analytecapture agent includes an analyte binding moiety and a capture agentbarcode domain.

FIG. 9 is a schematic diagram of an exemplary analyte capture agent 902comprised of an analyte binding moiety 904 and a capture agent barcodedomain 908. An analyte binding moiety 904 is a molecule capable ofbinding to an analyte 906 and interacting with a spatially-barcodedcapture probe. The analyte binding moiety can bind to the analyte 906with high affinity and/or with high specificity. The analyte captureagent can include a capture agent barcode domain 908, a nucleotidesequence (e.g., an oligonucleotide), which can hybridize to at least aportion or an entirety of a capture domain of a capture probe. Theanalyte binding moiety 904 can include a polypeptide and/or an aptamer(e.g., an oligonucleotide or peptide molecule that binds to a specifictarget analyte). The analyte binding moiety 904 can include an antibodyor antibody fragment (e.g., an antigen-binding fragment).

As used herein, the term “analyte binding moiety” refers to a moleculeor moiety capable of binding to a macromolecular constituent (e.g., ananalyte, e.g., a biological analyte). In some embodiments of any of thespatial profiling methods described herein, the analyte binding moietyof the analyte capture agent that binds to a biological analyte caninclude, but is not limited to, an antibody, or an epitope bindingfragment thereof, a cell surface receptor binding molecule, a receptorligand, a small molecule, a bi-specific antibody, a bi-specific T-cellengager, a T-cell receptor engager, a B-cell receptor engager, apro-body, an aptamer, a monobody, an affimer, a darpin, and a proteinscaffold, or any combination thereof. The analyte binding moiety canbind to the macromolecular constituent (e.g., analyte) with highaffinity and/or with high specificity. The analyte binding moiety caninclude a nucleotide sequence (e.g., an oligonucleotide), which cancorrespond to at least a portion or an entirety of the analyte bindingmoiety. The analyte binding moiety can include a polypeptide and/or anaptamer (e.g., a polypeptide and/or an aptamer that binds to a specifictarget molecule, e.g., an analyte). The analyte binding moiety caninclude an antibody or antibody fragment (e.g., an antigen-bindingfragment) that binds to a specific analyte (e.g., a polypeptide).

In some embodiments, an analyte binding moiety of an analyte captureagent includes one or more antibodies or antigen binding fragmentsthereof. The antibodies or antigen binding fragments including theanalyte binding moiety can specifically bind to a target analyte. Insome embodiments, the analyte is a protein (e.g., a protein on a surfaceof the biological sample (e.g., a cell) or an intracellular protein). Insome embodiments, a plurality of analyte capture agents comprising aplurality of analyte binding moieties bind a plurality of analytespresent in a biological sample. In some embodiments, the plurality ofanalytes includes a single species of analyte (e.g., a single species ofpolypeptide). In some embodiments in which the plurality of analytesincludes a single species of analyte, the analyte binding moieties ofthe plurality of analyte capture agents are the same. In someembodiments in which the plurality of analytes includes a single speciesof analyte, the analyte binding moieties of the plurality of analytecapture agents are the different (e.g., members of the plurality ofanalyte capture agents can have two or more species of analyte bindingmoieties, wherein each of the two or more species of analyte bindingmoieties binds a single species of analyte, e.g., at different bindingsites). In some embodiments, the plurality of analytes includes multipledifferent species of analyte (e.g., multiple different species ofpolypeptides).

An analyte capture agent can include an analyte binding moiety. Theanalyte binding moiety can be an antibody.

Further, exemplary, non-limiting antibodies that can be used as analytebinding moieties in an analyte capture agent or that can be used in theIHC/IF applications disclosed herein include any of the followingantibodies (and variations thereof) to: cell surface proteins,intracellular proteins, kinases, transcription factors, growth factorreceptors, tumor suppressors.

In some embodiments, analyte capture agents are capable of binding toanalytes present inside a cell. In some embodiments, analyte captureagents are capable of binding to cell surface analytes that can include,without limitation, a receptor, an antigen, a surface protein, atransmembrane protein, a cluster of differentiation protein, a proteinchannel, a protein pump, a carrier protein, a phospholipid, aglycoprotein, a glycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, an extracellular matrix protein, aposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation)state of a cell surface protein, a gap junction, and an adherensjunction. In some embodiments, the analyte capture agents are capable ofbinding to cell surface analytes that are post-translationally modified.In such embodiments, analyte capture agents can be specific for cellsurface analytes based on a given state of posttranslationalmodification (e.g., phosphorylation, glycosylation, ubiquitination,nitrosylation, methylation, acetylation or lipidation), such that a cellsurface analyte profile can include posttranslational modificationinformation of one or more analytes.

In some embodiments, the analyte capture agent includes a capture agentbarcode domain that is conjugated or otherwise attached to the analytebinding moiety. In some embodiments, the capture agent barcode domain iscovalently-linked to the analyte binding moiety. In some embodiments, acapture agent barcode domain is a nucleic acid sequence. In someembodiments, a capture agent barcode domain includes an analyte bindingmoiety barcode and an analyte capture sequence.

As used herein, the term “analyte binding moiety barcode” refers to abarcode that is associated with or otherwise identifies the analytebinding moiety. In some embodiments, by identifying an analyte bindingmoiety and its associated analyte binding moiety barcode, the analyte towhich the analyte binding moiety binds can also be identified. Ananalyte binding moiety barcode can be a nucleic acid sequence of a givenlength and/or sequence that is associated with the analyte bindingmoiety. An analyte binding moiety barcode can generally include any ofthe variety of aspects of barcodes described herein. For example, ananalyte capture agent that is specific to one type of analyte can havecoupled thereto a first capture agent barcode domain (e.g., thatincludes a first analyte binding moiety barcode), while an analytecapture agent that is specific to a different analyte can have adifferent capture agent barcode domain (e.g., that includes a secondbarcode analyte binding moiety barcode) coupled thereto. In someaspects, such a capture agent barcode domain can include an analytebinding moiety barcode that permits identification of the analytebinding moiety to which the capture agent barcode domain is coupled. Theselection of the capture agent barcode domain can allow significantdiversity in terms of sequence, while also being readily attachable tomost analyte binding moieties (e.g., antibodies or aptamers) as well asbeing readily detected, (e.g., using sequencing or array technologies).

In some embodiments, the capture agent barcode domain of an analytecapture agent includes an analyte capture sequence. As used herein, theterm “analyte capture sequence” refers to a region or moiety configuredto hybridize to, bind to, couple to, or otherwise interact with acapture domain of a capture probe. In some embodiments, an analytecapture sequence includes a nucleic acid sequence that is complementaryto or substantially complementary to the capture domain of a captureprobe such that the analyte capture sequence hybridizes to the capturedomain of the capture probe. In some embodiments, an analyte capturesequence comprises a poly(A) nucleic acid sequence that hybridizes to acapture domain that comprises a poly(T) nucleic acid sequence. In someembodiments, an analyte capture sequence comprises a poly(T) nucleicacid sequence that hybridizes to a capture domain that comprises apoly(A) nucleic acid sequence. In some embodiments, an analyte capturesequence comprises a non-homopolymeric nucleic acid sequence thathybridizes to a capture domain that comprises a non-homopolymericnucleic acid sequence that is complementary (or substantiallycomplementary) to the non-homopolymeric nucleic acid sequence of theanalyte capture region.

In some embodiments of any of the spatial analysis methods describedherein that employ an analyte capture agent, the capture agent barcodedomain can be directly coupled to the analyte binding moiety, or theycan be attached to a bead, molecular lattice, e.g., a linear, globular,cross-slinked, or other polymer, or other framework that is attached orotherwise associated with the analyte binding moiety, which allowsattachment of multiple capture agent barcode domains to a single analytebinding moiety. Attachment (coupling) of the capture agent barcodedomains to the analyte binding moieties can be achieved through any of avariety of direct or indirect, covalent or non-covalent associations orattachments. For example, in the case of a capture agent barcode domaincoupled to an analyte binding moiety that includes an antibody orantigen-binding fragment, such capture agent barcode domains can becovalently attached to a portion of the antibody or antigen-bindingfragment using chemical conjugation techniques (e.g., Lightning-Link®antibody labelling kits available from Innova Biosciences). In someembodiments, a capture agent barcode domain can be coupled to anantibody or antigen-binding fragment using non-covalent attachmentmechanisms (e.g., using biotinylated antibodies and oligonucleotides orbeads that include one or more biotinylated linker(s), coupled tooligonucleotides with an avidin or streptavidin linker.) Antibody andoligonucleotide biotinylation techniques can be used, and are describedfor example in Fang et al., Nucleic Acids Res. (2003), 31(2): 708-715,the entire contents of which are incorporated by reference herein.Likewise, protein and peptide biotinylation techniques have beendeveloped and can be used, and are described for example in U.S. Pat.No. 6,265,552, the entire contents of which are incorporated byreference herein. Furthermore, click reaction chemistry such as amethyltetrazine-PEG5-NHS ester reaction, a TCO-PEG4-NHS ester reaction,or the like, can be used to couple capture agent barcode domains toanalyte binding moieties. The reactive moiety on the analyte bindingmoiety can also include amine for targeting aldehydes, amine fortargeting maleimide (e.g., free thiols), azide for targeting clickchemistry compounds (e.g., alkynes), biotin for targeting streptavidin,phosphates for targeting EDC, which in turn targets active ester (e.g.,NH2). The reactive moiety on the analyte binding moiety can be achemical compound or group that binds to the reactive moiety on theanalyte binding moiety. Exemplary strategies to conjugate the analytebinding moiety to the capture agent barcode domain include the use ofcommercial kits (e.g., Solulink, Thunder link), conjugation of mildreduction of hinge region and maleimide labelling, stain-promoted clickchemistry reaction to labeled amides (e.g., copper-free), andconjugation of periodate oxidation of sugar chain and amine conjugation.In the cases where the analyte binding moiety is an antibody, theantibody can be modified prior to or contemporaneously with conjugationof the oligonucleotide. For example, the antibody can be glycosylatedwith a chemical substrate-permissive mutant ofβ-1,4-galactosyltransferase, GalT (Y289L) and azide-bearing uridinediphosphate-N-acetylgalactosamine analog uridine diphosphate-GalNAz. Themodified antibody can be conjugated to an oligonucleotide with adibenzocyclooctyne-PEG4-NHS group. In some embodiments, certain steps(e.g., COOH activation (e.g., EDC) and homobifunctional cross linkers)can be avoided to prevent the analyte binding moieties from conjugatingto themselves. In some embodiments of any of the spatial profilingmethods described herein, the analyte capture agent (e.g., analytebinding moiety coupled to an oligonucleotide) can be delivered into thecell, e.g., by transfection (e.g., using transfectamine, cationicpolymers, calcium phosphate or electroporation), by transduction (e.g.,using a bacteriophage or recombinant viral vector), by mechanicaldelivery (e.g., magnetic beads), by lipid (e.g.,1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)), or by transporterproteins. An analyte capture agent can be delivered into a cell usingexosomes. For example, a first cell can be generated that releasesexosomes comprising an analyte capture agent. An analyte capture agentcan be attached to an exosome membrane. An analyte capture agent can becontained within the cytosol of an exosome. Released exosomes can beharvested and provided to a second cell, thereby delivering the analytecapture agent into the second cell. An analyte capture agent can bereleasable from an exosome membrane before, during, or after deliveryinto a cell. In some embodiments, the cell is permeabilized to allow theanalyte capture agent to couple with intracellular constituents (suchas, without limitation, intracellular proteins, metabolites, and nuclearmembrane proteins). Following intracellular delivery, analyte captureagents can be used to analyze intracellular constituents as describedherein.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domain coupled to an analyte captureagent can include modifications that render it non-extendable by apolymerase. In some embodiments, when binding to a capture domain of acapture probe or nucleic acid in a sample for a primer extensionreaction, the capture agent barcode domain can serve as a template, nota primer. When the capture agent barcode domain also includes a barcode(e.g., an analyte binding moiety barcode), such a design can increasethe efficiency of molecular barcoding by increasing the affinity betweenthe capture agent barcode domain and unbarcoded sample nucleic acids,and eliminate the potential formation of adaptor artifacts. In someembodiments, the capture agent barcode domain can include a random N-mersequence that is capped with modifications that render it non-extendableby a polymerase. In some cases, the composition of the random N-mersequence can be designed to maximize the binding efficiency to free,unbarcoded ssDNA molecules. The design can include a random sequencecomposition with a higher GC content, a partial random sequence withfixed G or C at specific positions, the use of guanosines, the use oflocked nucleic acids, or any combination thereof.

A modification for blocking primer extension by a polymerase can be acarbon spacer group of different lengths or a dideoxynucleotide. In someembodiments, the modification can be an abasic site that has an apurineor apyrimidine structure, a base analog, or an analogue of a phosphatebackbone, such as a backbone of N-(2-aminoethyl)-glycine linked by amidebonds, tetrahydrofuran, or 1′, 2′-Dideoxyribose. The modification canalso be a uracil base, 2′OMe modified RNA, C3-18 spacers (e.g.,structures with 3-18 consecutive carbon atoms, such as C3 spacer),ethylene glycol multimer spacers (e.g., spacer 18 (hexa-ethyleneglycolspacer), biotin, di-deoxynucleotide triphosphate, ethylene glycol,amine, or phosphate.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domain coupled to the analyte bindingmoiety includes a cleavable domain. For example, after the analytecapture agent binds to an analyte (e.g., a cell surface analyte), thecapture agent barcode domain can be cleaved and collected for downstreamanalysis according to the methods as described herein. In someembodiments, the cleavable domain of the capture agent barcode domainincludes a U-excising element that allows the species to release fromthe bead. In some embodiments, the U-excising element can include asingle-stranded DNA (ssDNA) sequence that contains at least one uracil.The species can be attached to a bead via the ssDNA sequence. Thespecies can be released by a combination of uracil-DNA glycosylase(e.g., to remove the uracil) and an endonuclease (e.g., to induce anssDNA break). If the endonuclease generates a 5′ phosphate group fromthe cleavage, then additional enzyme treatment can be included indownstream processing to eliminate the phosphate group, e.g., prior toligation of additional sequencing handle elements, e.g., Illumina fullP5 sequence, partial P5 sequence, full R1 sequence, and/or partial R1sequence.

In some embodiments, multiple different species of analytes (e.g.,polypeptides) from the biological sample can be subsequently associatedwith the one or more physical properties of the biological sample. Forexample, the multiple different species of analytes can be associatedwith locations of the analytes in the biological sample. Suchinformation (e.g., proteomic information when the analyte bindingmoiety(ies) recognizes a polypeptide(s)) can be used in association withother spatial information (e.g., genetic information from the biologicalsample, such as DNA sequence information, transcriptome information(i.e., sequences of transcripts), or both). For example, a cell surfaceprotein of a cell can be associated with one or more physical propertiesof the cell (e.g., a shape, size, activity, or a type of the cell). Theone or more physical properties can be characterized by imaging thecell. The cell can be bound by an analyte capture agent comprising ananalyte binding moiety that binds to the cell surface protein and ananalyte binding moiety barcode that identifies that analyte bindingmoiety, and the cell can be subjected to spatial analysis (e.g., any ofthe variety of spatial analysis methods described herein). For example,the analyte capture agent bound to the cell surface protein can be boundto a capture probe (e.g., a capture probe on an array), which captureprobe includes a capture domain that interacts with an analyte capturesequence present on the capture agent barcode domain of the analytecapture agent. All or part of the capture agent barcode domain(including the analyte binding moiety barcode) can be copied with apolymerase using a 3′ end of the capture domain as a priming site,generating an extended capture probe that includes the all or part ofcomplementary sequence that corresponds to the capture probe (includinga spatial barcode present on the capture probe) and a copy of theanalyte binding moiety barcode. In some embodiments, an analyte captureagent with an extended capture agent barcode domain that includes asequence complementary to a spatial barcode of a capture probe is calleda “spatially-tagged analyte capture agent.”

In some embodiments, the spatial array with spatially-tagged analytecapture agents can be contacted with a sample, where the analyte captureagent(s) associated with the spatial array capture the targetanalyte(s). The analyte capture agent(s) containing the extended captureprobe(s), which includes a sequence complementary to the spatialbarcode(s) of the capture probe(s) and the analyte binding moietybarcode(s), can then be denatured from the capture probe(s) of thespatial array. This allows the spatial array to be reused. The samplecan be dissociated into non-aggregated cells (e.g., single cells) andanalyzed by the single cell/droplet methods described herein. Thespatially-tagged analyte capture agent can be sequenced to obtain thenucleic acid sequence of the spatial barcode of the capture probe andthe analyte binding moiety barcode of the analyte capture agent. Thenucleic acid sequence of the extended capture probe can thus beassociated with an analyte (e.g., cell surface protein), and in turn,with the one or more physical properties of the cell (e.g., a shape orcell type). In some embodiments, the nucleic acid sequence of theextended capture probe can be associated with an intracellular analyteof a nearby cell, where the intracellular analyte was released using anyof the cell permeabilization or analyte migration techniques describedherein.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domains released from the analytecapture agents can then be subjected to sequence analysis to identifywhich analyte capture agents were bound to analytes. Based upon thecapture agent barcode domains that are associated with a feature (e.g.,a feature at a particular location) on a spatial array and the presenceof the analyte binding moiety barcode sequence, an analyte profile canbe created for a biological sample. Profiles of individual cells orpopulations of cells can be compared to profiles from other cells, e.g.,‘normal’ cells, to identify variations in analytes, which can providediagnostically relevant information. In some embodiments, these profilescan be useful in the diagnosis of a variety of disorders that arecharacterized by variations in cell surface receptors, such as cancerand other disorders.

FIG. 10 is a schematic diagram depicting an exemplary interactionbetween a feature-immobilized capture probe 1024 and an analyte captureagent 1026. The feature-immobilized capture probe 1024 can include aspatial barcode 1008 as well as one or more functional sequences 1006and 1010, as described elsewhere herein. The capture probe can alsoinclude a capture domain 1012 that is capable of binding to an analytecapture agent 1026. The analyte capture agent 1026 can include afunctional sequence 1018, capture agent barcode domain 1016, and ananalyte capture sequence 1014 that is capable of binding to the capturedomain 1012 of the capture probe 1024. The analyte capture agent canalso include a linker 1020 that allows the capture agent barcode domain1016 to couple to the analyte binding moiety 1022.

In some embodiments of any of the spatial profiling methods describedherein, the methods are used to identify immune cell profiles. Immunecells express various adaptive immunological receptors relating toimmune function, such as T cell receptors (TCRs) and B cell receptors(BCRs). T cell receptors and B cell receptors play a part in the immuneresponse by specifically recognizing and binding to antigens and aidingin their destruction.

The T cell receptor, or TCR, is a molecule found on the surface of Tcells that is generally responsible for recognizing fragments of antigenas peptides bound to major histocompatibility complex (MHC) molecules.The TCR is generally a heterodimer of two chains, each of which is amember of the immunoglobulin superfamily, possessing an N-terminalvariable (V) domain, and a C terminal constant domain. In humans, in 95%of T cells, the TCR consists of an alpha (α) and beta (β) chain, whereasin 5% of T cells, the TCR consists of gamma and delta (γ/δ) chains. Thisratio can change during ontogeny and in diseased states as well as indifferent species. When the TCR engages with antigenic peptide and MHC(peptide/MHC or pMHC), the T lymphocyte is activated through signaltransduction.

Each of the two chains of a TCR contains multiple copies of genesegments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, anda joining ‘J’ gene segment. The TCR alpha chain (TCRa) is generated byrecombination of V and J segments, while the beta chain (TCRb) isgenerated by recombination of V, D, and J segments. Similarly,generation of the TCR gamma chain involves recombination of V and J genesegments, while generation of the TCR delta chain occurs byrecombination of V, D, and J gene segments. The intersection of thesespecific regions (V and J for the alpha or gamma chain, or V, D and Jfor the beta or delta chain) corresponds to the CDR3 region that isimportant for antigen-MHC recognition. Complementarity determiningregions (e.g., CDR1, CDR2, and CDR3), or hypervariable regions, aresequences in the variable domains of antigen receptors (e.g., T cellreceptor and immunoglobulin) that can complement an antigen. Most of thediversity of CDRs is found in CDR3, with the diversity being generatedby somatic recombination events during the development of T lymphocytes.A unique nucleotide sequence that arises during the gene arrangementprocess can be referred to as a clonotype.

The B cell receptor, or BCR, is a molecule found on the surface of Bcells. The antigen binding portion of a BCR is composed of amembrane-bound antibody that, like most antibodies (e.g.,immunoglobulins), has a unique and randomly determined antigen-bindingsite. The antigen binding portion of a BCR includes membrane-boundimmunoglobulin molecule of one isotype (e.g., IgD, IgM, IgA, IgG, orIgE). When a B cell is activated by its first encounter with a cognateantigen, the cell proliferates and differentiates to generate apopulation of antibody-secreting plasma B cells and memory B cells. Thevarious immunoglobulin isotypes differ in their biological features,structure, target specificity, and distribution. A variety of molecularmechanisms exist to generate initial diversity, including geneticrecombination at multiple sites.

The BCR is composed of two genes IgH and IgK (or IgL) coding forantibody heavy and light chains. Immunoglobulins are formed byrecombination among gene segments, sequence diversification at thejunctions of these segments, and point mutations throughout the gene.Each heavy chain gene contains multiple copies of three different genesegments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, anda joining ‘J’ gene segment. Each light chain gene contains multiplecopies of two different gene segments for the variable region of theprotein—a variable ‘V’ gene segment and a joining ‘J’ gene segment.

The recombination can generate a molecule with one of each of the V, D,and J segments. Furthermore, several bases can be deleted and othersadded (called N and P nucleotides) at each of the two junctions, therebygenerating further diversity. After B cell activation, a process ofaffinity maturation through somatic hypermutation occurs. In thisprocess, progeny cells of the activated B cells accumulate distinctsomatic mutations throughout the gene with higher mutation concentrationin the CDR regions leading to the generation of antibodies with higheraffinity to the antigens.

In addition to somatic hypermutation, activated B cells undergo theprocess of isotype switching. Antibodies with the same variable segmentscan have different forms (isotypes) depending on the constant segment.Whereas all naïve B cells express IgM (or IgD), activated B cells mostlyexpress IgG but also IgM, IgA, and IgE. This expression switching fromIgM (and/or IgD) to IgG, IgA, or IgE occurs through a recombinationevent causing one cell to specialize in producing a specific isotype. Aunique nucleotide sequence that arises during the gene arrangementprocess can similarly be referred to as a clonotype.

Certain methods described herein are utilized to analyze the varioussequences of TCRs and BCRs from immune cells, for example, variousclonotypes. In some embodiments, the methods are used to analyze thesequence of a TCR alpha chain, a TCR beta chain, a TCR delta chain, aTCR gamma chain, or any fragment thereof (e.g., variable regionsincluding V(D)J or VJ regions, constant regions, transmembrane regions,fragments thereof, combinations thereof, and combinations of fragmentsthereof). In some embodiments, the methods described herein can be usedto analyze the sequence of a B cell receptor heavy chain, B cellreceptor light chain, or any fragment thereof (e.g., variable regionsincluding V(D)J or VJ regions, constant regions, transmembrane regions,fragments thereof, combinations thereof, and combinations of fragmentsthereof).

Where immune cells are to be analyzed, primer sequences useful in any ofthe various operations for attaching barcode sequences and/oramplification reactions can include gene specific sequences which targetgenes or regions of genes of immune cell proteins, for example immunereceptors. Such gene sequences include, but are not limited to,sequences of various T cell receptor alpha variable genes (TRAV genes),T cell receptor alpha joining genes (TRAJ genes), T cell receptor alphaconstant genes (TRAC genes), T cell receptor beta variable genes (TRBVgenes), T cell receptor beta diversity genes (TRBD genes), T cellreceptor beta joining genes (TRBJ genes), T cell receptor beta constantgenes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), Tcell receptor gamma joining genes (TRGJ genes), T cell receptor gammaconstant genes (TRGC genes), T cell receptor delta variable genes (TRDVgenes), T cell receptor delta diversity genes (TRDD genes), T cellreceptor delta joining genes (TRDJ genes), and T cell receptor deltaconstant genes (TRDC genes).

In some embodiments, the analyte binding moiety is based on the MajorHistocompatibility Complex (MHC) class I or class II. In someembodiments, the analyte binding moiety is an MHC multimer including,without limitation, MHC dextramers, MHC tetramers, and MHC pentamers(see, for example, U.S. Patent Application Publication Nos. US2018/0180601 and US 2017/0343545, the entire contents of each of whichare incorporated herein by reference. MHCs (e.g., a soluble MHC monomermolecule), including full or partial MHC-peptides, can be used asanalyte binding moieties of analyte capture agents that are coupled tocapture agent barcode domains that include an analyte binding moietybarcode that identifies its associated MHC (and, thus, for example, theMHC's TCR binding partner). In some embodiments, MHCs are used toanalyze one or more cell-surface features of a T-cell, such as a TCR. Insome cases, multiple MHCs are associated together in a larger complex(MHC multi-mer) to improve binding affinity of MHCs to TCRs via multipleligand binding synergies.

FIGS. 11A, 11B, and 11C are schematics illustrating how streptavidincell tags can be utilized in an array-based system to produce aspatially-barcoded cell or cellular contents. For example, as shown inFIG. 11A, peptide-bound major histocompatibility complex (MHC) can beindividually associated with biotin (β2m) and bound to a streptavidinmoiety such that the streptavidin moiety comprises multiple pMHCmoieties. Each of these moieties can bind to a TCR such that thestreptavidin binds to a target T-cell via multiple MCH/TCR bindinginteractions. Multiple interactions synergize and can substantiallyimprove binding affinity. Such improved affinity can improve labellingof T-cells and also reduce the likelihood that labels will dissociatefrom T-cell surfaces. As shown in FIG. 11B, a capture agent barcodedomain 1101 can be modified with streptavidin 1102 and contacted withmultiple molecules of biotinylated MHC 1103 such that the biotinylatedMHC 1103 molecules are coupled with the streptavidin conjugated captureagent barcode domain 1101. The result is a barcoded MHC multimer complex1105. As shown in FIG. 11B, the capture agent barcode domain sequence1101 can identify the MHC as its associated label and also includesoptional functional sequences such as sequences for hybridization withother oligonucleotides. As shown in FIG. 11C, one exampleoligonucleotide is capture probe 1106 that comprises a complementarysequence (e.g., rGrGrG corresponding to C C C), a barcode sequence andother functional sequences, such as, for example, a UMI, an adaptersequence (e.g., comprising a sequencing primer sequence (e.g., R1 or apartial R1 (“pR1”), R2), a flow cell attachment sequence (e.g., P5 or P7or partial sequences thereof)), etc. In some cases, capture probe 1106may at first be associated with a feature (e.g., a gel bead) andreleased from the feature. In other embodiments, capture probe 1106 canhybridize with a capture agent barcode domain 1101 of theMHC-oligonucleotide complex 1105. The hybridized oligonucleotides(Spacer C C C and Spacer rGrGrG) can then be extended in primerextension reactions such that constructs comprising sequences thatcorrespond to each of the two spatial barcode sequences (the spatialbarcode associated with the capture probe, and the barcode associatedwith the MHC-oligonucleotide complex) are generated. In some cases, oneor both of these corresponding sequences may be a complement of theoriginal sequence in capture probe 1106 or capture agent barcode domain1101. In other embodiments, the capture probe and the capture agentbarcode domain are ligated together. The resulting constructs can beoptionally further processed (e.g., to add any additional sequencesand/or for clean-up) and subjected to sequencing. As described elsewhereherein, a sequence derived from the capture probe 1106 spatial barcodesequence may be used to identify a feature and the sequence derived fromspatial barcode sequence on the capture agent barcode domain 1101 may beused to identify the particular peptide MHC complex 1104 bound on thesurface of the cell (e.g., when using MHC-peptide libraries forscreening immune cells or immune cell populations).

(c) Substrates

For the spatial array-based analytical methods described herein, asubstrate functions as a support for direct or indirect attachment ofcapture probes to features of the array. In addition, in someembodiments, a substrate (e.g., the same substrate or a differentsubstrate) can be used to provide support to a biological sample,particularly, for example, a thin tissue section. Accordingly, a“substrate” is a support that is insoluble in aqueous liquid and whichallows for positioning of biological samples, analytes, features, and/orcapture probes on the substrate.

Further, a “substrate” as used herein, and when not preceded by themodifier “chemical”, refers to a member with at least one surface thatgenerally functions to provide physical support for biological samples,analytes, and/or any of the other chemical and/or physical moieties,agents, and structures described herein. Substrates can be formed from avariety of solid materials, gel-based materials, colloidal materials,semi-solid materials (e.g., materials that are at least partiallycross-linked), materials that are fully or partially cured, andmaterials that undergo a phase change or transition to provide physicalsupport. Examples of substrates that can be used in the methods andsystems described herein include, but are not limited to, slides (e.g.,slides formed from various glasses, slides formed from variouspolymers), hydrogels, layers and/or films, membranes (e.g., porousmembranes), flow cells, cuvettes, wafers, plates, or combinationsthereof. In some embodiments, substrates can optionally includefunctional elements such as recesses, protruding structures,microfluidic elements (e.g., channels, reservoirs, electrodes, valves,seals), and various markings, as will be discussed in further detailbelow.

(i) Substrate Attributes

A substrate can generally have any suitable form or format. For example,a substrate can be flat, curved, e.g., convexly or concavely curvedtowards the area where the interaction between a biological sample,e.g., tissue sample, and a substrate takes place. In some embodiments, asubstrate is flat, e.g., planar, chip, or slide. A substrate can containone or more patterned surfaces within the substrate (e.g., channels,wells, projections, ridges, divots, etc.).

A substrate can be of any desired shape. For example, a substrate can betypically a thin, flat shape (e.g., a square or a rectangle). In someembodiments, a substrate structure has rounded corners (e.g., forincreased safety or robustness). In some embodiments, a substratestructure has one or more cut-off corners (e.g., for use with a slideclamp or cross-table). In some embodiments, where a substrate structureis flat, the substrate structure can be any appropriate type of supporthaving a flat surface (e.g., a chip or a slide such as a microscopeslide).

Substrates can optionally include various structures such as, but notlimited to, projections, ridges, and channels. A substrate can bemicropatterned to limit lateral diffusion (e.g., to prevent overlap ofspatial barcodes). A substrate modified with such structures can bemodified to allow association of analytes, features (e.g., beads), orprobes at individual sites. For example, the sites where a substrate ismodified with various structures can be contiguous or non-contiguouswith other sites.

In some embodiments, the surface of a substrate can be modified so thatdiscrete sites are formed that can only have or accommodate a singlefeature. In some embodiments, the surface of a substrate can be modifiedso that features adhere to random sites.

In some embodiments, the surface of a substrate is modified to containone or more wells, using techniques such as (but not limited to)stamping, microetching, or molding techniques. In some embodiments inwhich a substrate includes one or more wells, the substrate can be aconcavity slide or cavity slide. For example, wells can be formed by oneor more shallow depressions on the surface of the substrate. In someembodiments, where a substrate includes one or more wells, the wells canbe formed by attaching a cassette (e.g., a cassette containing one ormore chambers) to a surface of the substrate structure.

In some embodiments, the structures of a substrate (e.g., wells orfeatures) can each bear a different capture probe. Different captureprobes attached to each structure can be identified according to thelocations of the structures in or on the surface of the substrate.Exemplary substrates include arrays in which separate structures arelocated on the substrate including, for example, those having wells thataccommodate features.

In some embodiments where the substrate is modified to contain one ormore structures, including but not limited to, wells, projections,ridges, features, or markings, the structures can include physicallyaltered sites. For example, a substrate modified with various structurescan include physical properties, including, but not limited to, physicalconfigurations, magnetic or compressive forces, chemicallyfunctionalized sites, chemically altered sites, and/or electrostaticallyaltered sites. In some embodiments where the substrate is modified tocontain various structures, including but not limited to wells,projections, ridges, features, or markings, the structures are appliedin a pattern. Alternatively, the structures can be randomly distributed.

The substrate (e.g., or a bead or a feature on an array) can includetens to hundreds of thousands or millions of individual oligonucleotidemolecules (e.g., at least about 10,000, 50,000, 100,000, 500,000,1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000oligonucleotide molecules).

In some embodiments, a substrate includes one or more markings on asurface of a substrate, e.g., to provide guidance for correlatingspatial information with the characterization of the analyte ofinterest. For example, a substrate can be marked with a grid of lines(e.g., to allow the size of objects seen under magnification to beeasily estimated and/or to provide reference areas for countingobjects). In some embodiments, fiducial markers can be included on asubstrate. Such markings can be made using techniques including, but notlimited to, printing, sand-blasting, and depositing on the surface.

In some embodiments, imaging can be performed using one or more fiducialmarkers, i.e., objects placed in the field of view of an imaging systemwhich appear in the image produced. Fiducial markers are typically usedas a point of reference or measurement scale. Fiducial markers caninclude, but are not limited to, detectable labels such as fluorescent,radioactive, chemiluminescent, and colorimetric labels. The use offiducial markers to stabilize and orient biological samples isdescribed, for example, in Carter et al., Applied Optics 46:421-427,2007), the entire contents of which are incorporated herein byreference. In some embodiments, a fiducial marker can be a physicalparticle (e.g., a nanoparticle, a microsphere, a nanosphere, a bead, apost, or any of the other exemplary physical particles described hereinor known in the art).

In some embodiments, a fiducial marker can be present on a substrate toprovide orientation of the biological sample. In some embodiments, amicrosphere can be coupled to a substrate to aid in orientation of thebiological sample. In some examples, a microsphere coupled to asubstrate can produce an optical signal (e.g., fluorescence). In anotherexample, a microsphere can be attached to a portion (e.g., corner) of anarray in a specific pattern or design (e.g., hexagonal design) to aid inorientation of a biological sample on an array of features on thesubstrate. In some embodiments, a quantum dot can be coupled to thesubstrate to aid in the orientation of the biological sample. In someexamples, a quantum dot coupled to a substrate can produce an opticalsignal.

In some embodiments, a fiducial marker can be an immobilized moleculewith which a detectable signal molecule can interact to generate asignal. For example, a marker nucleic acid can be linked or coupled to achemical moiety capable of fluorescing when subjected to light of aspecific wavelength (or range of wavelengths). Such a marker nucleicacid molecule can be contacted with an array before, contemporaneouslywith, or after the tissue sample is stained to visualize or image thetissue section. Although not required, it can be advantageous to use amarker that can be detected using the same conditions (e.g., imagingconditions) used to detect a labelled cDNA.

In some embodiments, fiducial markers are included to facilitate theorientation of a tissue sample or an image thereof in relation to animmobilized capture probes on a substrate. Any number of methods formarking an array can be used such that a marker is detectable only whena tissue section is imaged. For instance, a molecule, e.g., afluorescent molecule that generates a signal, can be immobilizeddirectly or indirectly on the surface of a substrate. Markers can beprovided on a substrate in a pattern (e.g., an edge, one or more rows,one or more lines, etc.).

In some embodiments, a fiducial marker can be randomly placed in thefield of view. For example, an oligonucleotide containing a fluorophorecan be randomly printed, stamped, synthesized, or attached to asubstrate (e.g., a glass slide) at a random position on the substrate. Atissue section can be contacted with the substrate such that theoligonucleotide containing the fluorophore contacts, or is in proximityto, a cell from the tissue section or a component of the cell (e.g., anmRNA or DNA molecule). An image of the substrate and the tissue sectioncan be obtained, and the position of the fluorophore within the tissuesection image can be determined (e.g., by reviewing an optical image ofthe tissue section overlaid with the fluorophore detection). In someembodiments, fiducial markers can be precisely placed in the field ofview (e.g., at known locations on a substrate). In this instance, afiducial marker can be stamped, attached, or synthesized on thesubstrate and contacted with a biological sample. Typically, an image ofthe sample and the fiducial marker is taken, and the position of thefiducial marker on the substrate can be confirmed by viewing the image.

In some embodiments, a fiducial marker can be an immobilized molecule(e.g., a physical particle) attached to the substrate. For example, afiducial marker can be a nanoparticle, e.g., a nanorod, a nanowire, ananocube, a nanopyramid, or a spherical nanoparticle. In some examples,the nanoparticle can be made of a heavy metal (e.g., gold). In someembodiments, the nanoparticle can be made from diamond. In someembodiments, the fiducial marker can be visible by eye.

As noted herein, any of the fiducial markers described herein (e.g.,microspheres, beads, or any of the other physical particles describedherein) can be located at a portion (e.g., corner) of an array in aspecific pattern or design (e.g., hexagonal design) to aid inorientation of a biological sample on an array of features on thesubstrate. In some embodiments, the fiducial markers located at aportion (e.g., corner) of an array (e.g., an array on a substrate) canbe patterned or designed in at least 1, at least 2, at least 3, or atleast 4 unique patterns. In some examples, the fiducial markers locatedat the corners of the array (e.g., an array on a substrate) can havefour unique patterns of fiducial markers.

In some examples, fiducial markers can surround the array. In someembodiments the fiducial markers allow for detection of, e.g.,mirroring. In some embodiments, the fiducial markers may completelysurround the array. In some embodiments, the fiducial markers may notcompletely surround the array. In some embodiments, the fiducial markersidentify the corners of the array. In some embodiments, one or morefiducial markers identify the center of the array. In some embodiments,the fiducial markers comprise patterned spots, wherein the diameter ofone or more patterned spot fiducial markers is approximately 100micrometers. The diameter of the fiducial markers can be any usefuldiameter including, but not limited to, 50 micrometers to 500micrometers in diameter. The fiducial markers may be arranged in such away that the center of one fiducial marker is between 100 micrometersand 200 micrometers from the center of one or more other fiducialmarkers surrounding the array. In some embodiments, the array with thesurrounding fiducial markers is approximately 8 mm by 8 mm. In someembodiments, the array without the surrounding fiducial markers issmaller than 8 mm by 50 mm.

In some embodiments, an array can be enclosed within a frame. Putanother way, the perimeter of an array can have fiducial markers suchthat the array is enclosed, or substantially enclosed. In someembodiments, the perimeter of an array can be fiducial markers (e.g.,any fiducial marker described herein). In some embodiments, theperimeter of an array can be uniform. For example, the fiducial markingscan connect, or substantially connect, consecutive corners of an arrayin such a fashion that the non-corner portion of the array perimeter isthe same on all sides (e.g., four sides) of the array. In someembodiments, the fiducial markers attached to the non-corner portions ofthe perimeter can be pattered or designed to aid in the orientation ofthe biological sample on the array. In some embodiments, the particlesattached to the non-corner portions of the perimeter can be patterned ordesigned in at least 1, at least 2, at least 3, or at least 4 patterns.In some embodiments, the patterns can have at least 2, at least 3, or atleast 4 unique patterns of fiducial markings on the non-corner portionof the array perimeter.

In some embodiments, an array can include at least two fiducial markers(e.g., at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 12, at least 15, at least 20,at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100 fiducial markers or more (e.g.,several hundred, several thousand, or tens of thousands of fiducialmarkers)) in distinct positions on the surface of a substrate. Fiducialmarkers can be provided on a substrate in a pattern (e.g., an edge, oneor more rows, one or more lines, etc.).

A wide variety of different substrates can be used for the foregoingpurposes. In general, a substrate can be any suitable support material.Exemplary substrates include, but are not limited to, glass, modifiedand/or functionalized glass, hydrogels, films, membranes, plastics(including e.g., acrylics, polystyrene, copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins,Zeonor, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, optical fiber bundles, andpolymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclicolefin polymers (COPs), polypropylene, polyethylene polycarbonate, orcombinations thereof.

Among the examples of substrate materials discussed above, polystyreneis a hydrophobic material suitable for binding negatively chargedmacromolecules because it normally contains few hydrophilic groups. Fornucleic acids immobilized on glass slides, by increasing thehydrophobicity of the glass surface the nucleic acid immobilization canbe increased. Such an enhancement can permit a relatively more denselypacked formation (e.g., provide improved specificity and resolution).

In another example, a substrate can be a flow cell. Flow cells can beformed of any of the foregoing materials, and can include channels thatpermit reagents, solvents, features, and analytes to pass through theflow cell. In some embodiments, a hydrogel embedded biological sample isassembled in a flow cell (e.g., the flow cell is utilized to introducethe hydrogel to the biological sample). In some embodiments, a hydrogelembedded biological sample is not assembled in a flow cell. In someembodiments, the hydrogel embedded biological sample can then beprepared and/or isometrically expanded as described herein.

(ii) Coatings

In some embodiments, a surface of a substrate can be coated with acell-permissive coating to allow adherence of live cells. A“cell-permissive coating” is a coating that allows or helps cells tomaintain cell viability (e.g., remain viable) on the substrate. Forexample, a cell-permissive coating can enhance cell attachment, cellgrowth, and/or cell differentiation, e.g., a cell-permissive coating canprovide nutrients to the live cells. A cell-permissive coating caninclude a biological material and/or a synthetic material. Acell-permissive coating can be used to culture adherent cells on aspatially-barcoded array, or to maintain cell viability of a tissuesample or section while in contact with a spatially-barcoded array.

In some embodiments, a substrate is coated with a surface treatment suchas poly(L)-lysine. Additionally or alternatively, the substrate can betreated by silanation, e.g., with epoxy-silane, amino-silane, and/or bya treatment with polyacrylamide.

In some embodiments, a substrate is treated in order to minimize orreduce non-specific analyte hybridization within or between features.For example, treatment can include coating the substrate with ahydrogel, film, and/or membrane that creates a physical barrier tonon-specific hybridization. Any suitable hydrogel can be used. Forexample, hydrogel matrices prepared according to the methods set forthin U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. PatentApplication Publication Nos. U.S. 2017/0253918 and U.S. 2018/0052081,can be used. The entire contents of each of the foregoing documents isincorporated herein by reference.

Treatment can include adding a functional group that is reactive orcapable of being activated such that it becomes reactive afterapplication of a stimulus (e.g., photoreactive functional groups).Treatment can include treating with polymers having one or more physicalproperties (e.g., mechanical, electrical, magnetic, and/or thermal) thatminimize non-specific binding (e.g., that activate a substrate atcertain locations to allow analyte hybridization at those locations).

A “removable coating” is a coating that can be removed from the surfaceof a substrate upon application of a releasing agent. In someembodiments, a removable coating includes a hydrogel as describedherein, e.g., a hydrogel including a polypeptide-based material.

In some embodiments, the hydrogel in the removable coating is astimulus-responsive hydrogel. A stimulus-responsive hydrogel can undergoa gel-to-solution and/or gel-to-solid transition upon application of oneor more external triggers (e.g., a releasing agent). See, e.g., Willner,Acc. Chem. Res. 50:657-658, 2017, which is incorporated herein byreference in its entirety. Non-limiting examples of astimulus-responsive hydrogel include a thermoresponsive hydrogel, apH-responsive hydrogel, a light-responsive hydrogel, a redox-responsivehydrogel, an analyte-responsive hydrogel, or a combination thereof. Insome embodiments, a stimulus-responsive hydrogel can be amulti-stimuli-responsive hydrogel.

A “releasing agent” or “external trigger” is an agent that allows forthe removal of a removable coating from a substrate when the releasingagent is applied to the removable coating. An external trigger orreleasing agent can include physical triggers such as thermal, magnetic,ultrasonic, electrochemical, and/or light stimuli as well as chemicaltriggers such as pH, redox reactions, supramolecular complexes, and/orbiocatalytically driven reactions. See e.g., Echeverria, et al., Gels(2018), 4, 54; doi:10.3390/gels4020054, which is incorporated herein byreference in its entirety. The type of “releasing agent” or “externaltrigger” can depend on the type of removable coating. For example, aremovable coating featuring a redox-responsive hydrogel can be removedupon application of a releasing agent that includes a reducing agentsuch as dithiothreitol (DTT). As another example, a pH-responsivehydrogel can be removed upon the application of a releasing agent thatchanges the pH.

(iii) Gel Substrates

In some embodiments, a hydrogel can form a substrate. The term“hydrogel” herein refers to a macromolecular polymer gel including anetwork. Within the network, some polymer chains can optionally becross-linked, although cross-linking does not always occur. In someembodiments, the substrate includes a hydrogel and one or more secondmaterials. In some embodiments, the hydrogel is placed on top of one ormore second materials. For example, the hydrogel can be pre-formed andthen placed on top of, underneath, or in any other configuration withone or more second materials. In some embodiments, hydrogel formationoccurs after contacting one or more second materials during formation ofthe substrate. Hydrogel formation can also occur within a structure(e.g., wells, ridges, features, projections, and/or markings) located ona substrate. Where the substrate includes a gel (e.g., a hydrogel or gelmatrix), oligonucleotides within the gel can attach to the substrate.

In some embodiments, a hydrogel can include hydrogel subunits.

In some embodiments, a hydrogel includes a hybrid material, e.g., thehydrogel material includes elements of both synthetic and naturalpolymers. Examples of suitable hydrogels are described, for example, inU.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. PatentApplication Publication Nos. 2017/0253918, 2018/0052081 and2010/0055733, the entire contents of each of which is incorporatedherein by reference.

In some embodiments, cross-linkers and/or initiators are added tohydrogel subunits. Examples of cross-linkers include, withoutlimitation, bis-acrylamide and diazirine. Examples of initiatorsinclude, without limitation, azobisisobutyronitrile (AIBN), riboflavin,and L-arginine. Inclusion of cross-linkers and/or initiators can lead toincreased covalent bonding between interacting biological macromoleculesin later polymerization steps.

In some embodiments, hydrogels can have a colloidal structure, such asagarose, or a polymer mesh structure, such as gelatin. In someembodiments, the hydrogel is a homopolymeric hydrogel. In someembodiments, the hydrogel is a copolymeric hydrogel. In someembodiments, the hydrogel is a multipolymer interpenetrating polymerichydrogel.

In some embodiments, some hydrogel subunits are polymerized (e.g.,undergo “formation”) covalently or physically cross-linked, to form ahydrogel network. For example, hydrogel subunits can be polymerized byany method including, but not limited to, thermal crosslinking, chemicalcrosslinking, physical crosslinking, ionic crosslinking,photo-crosslinking, free-radical initiation crosslinking, an additionreaction, condensation reaction, water-soluble crosslinking reactions,irradiative crosslinking (e.g., x-ray, electron beam), or combinationsthereof. Techniques such as lithographic photopolymerization can also beused to form hydrogels.

In some embodiments, gel beads containing oligonucleotides (e.g.,barcoded oligonucleotides such as capture probes) can be deposited on asubstrate (e.g., a glass slide). In some embodiments, gel pads can bedeposited on a substrate (e.g., a glass slide). In some embodiments, gelpads or gel beads are deposited on a substrate in an arrayed format. Insome embodiments in which gel pads or gel beads are deposited on asubstrate in an arrayed format, a hydrogel molecular precursor solutioncan be applied on top of the array (e.g., the array of gel pads or gelbeads on a glass slide). In some embodiments, a hydrogel molecularprecursor solution can be polymerized such that the deposited gel padsor gel beads are immobilized within the polymerized hydrogel. Anysuitable method of polymerization can be used or (e.g., any of thevariety of methods described herein). In some embodiments, a polymerizedhydrogel that includes the gel pads or gel beads can be removed (e.g.,peeled) from the substrate (e.g., glass slide) such that the gel beadsor gel pads are secured in the hydrogel. In some embodiments, apolymerized hydrogel that includes the gel pads or gel beads is aconductive substrate (as described herein) that can be used inaccordance with any of the variety of analyte capture methods describedherein (e.g., electrophoretic migration of analytes for capture).

Arrays can be prepared by depositing features (e.g., droplets, beads) ona substrate surface to produce a spatially-barcoded array. Methods ofdepositing (e.g., droplet manipulation) features are known in the art(see, U.S. Patent Application Publication No. 2008/0132429, Rubina, A.Y., et al., Biotechniques. 2003 May; 34(5):1008-14, 1016-20, 1022 andVasiliskov et al. Biotechniques. 1999 September; 27(3):592-4, 596-8, 600passim. each herein incorporated by reference in its entirety). Afeature can be printed or deposited at a specific location on thesubstrate (e.g., inkjet printing). In some embodiments, each feature canhave a unique oligonucleotide that functions as a spatial barcode. Insome embodiments, each feature can have capture probes for multiplexing(e.g., capturing multiple analytes or multiple types of analytes, e.g.,proteins and nucleic acids). In some embodiments, a feature can beprinted or deposited at the specific location using an electric field. Afeature can contain a photo-crosslinkable polymer precursor and anoligonucleotide. In some embodiments, the photo-crosslinkable polymerprecursor can be deposited into a patterned feature on the substrate(e.g., well).

A “photo-crosslinkable polymer precursor” refers to a compound thatcross-links and/or polymerizes upon exposure to light. In someembodiments, one or more photoinitiators may also be included to induceand/or promote polymerization and/or cross-linking. See, e.g., Choi etal. Biotechniques. 2019 January; 66(1):40-53, which is incorporatedherein by reference in its entirety.

Non-limiting examples of photo-crosslinkable polymer precursors includepolyethylene (glycol) diacrylate (PEGDA), gelatin-methacryloyl (GelMA),and methacrylated hyaluronic acid (MeHA). In some embodiments, aphoto-crosslinkable polymer precursor comprises polyethylene (glycol)diacrylate (PEGDA), gelatin-methacryloyl (GelMA), methacrylatedhyaluronic acid (MeHA), or a combination thereof. In some embodiments, aphoto-crosslinkable polymer precursor (e.g., PAZAM) can be covalentlylinked (e.g., cross-linked) to a substrate. In some embodiments, aphoto-crosslinkable polymer precursor is not covalently linked to asubstrate surface. For example, a silane-free acrylamide can be used(See U.S. Patent Application Publication No. 2011/0059865, hereinincorporated by reference in its entirety). The photo-crosslinkablepolymer precursor in a feature (e.g., droplet or bead) can bepolymerized by any known method. The oligonucleotides can be polymerizedin a cross-linked gel matrix (e.g., copolymerized or simultaneouslypolymerized). In some embodiments, the features containing thephoto-crosslinkable polymer precursor deposited on the substrate surfacecan be exposed to UV light. The UV light can induce polymerization ofthe photo-crosslinkable polymer precursor and result in the featuresbecoming a gel matrix (e.g., gel pads) on the substrate surface (e.g.,array).

Polymerization methods for hydrogel subunits can be selected to formhydrogels with different properties (e.g., pore volume, swellingproperties, biodegradability, conduction, transparency, and/orpermeability of the hydrogel). For example, a hydrogel can include poresof sufficient volume to allow the passage of macromolecules, (e.g.,nucleic acids, proteins, chromatin, metabolites, gRNA, antibodies,carbohydrates, peptides, metabolites, and/or small molecules) to/fromthe sample (e.g., tissue section). It is known that pore volumegenerally decreases with increasing concentration of hydrogel subunitsand generally increases with an increasing ratio of hydrogel subunits tocross-linker. Therefore, a hydrogel composition can be prepared thatincludes a concentration of hydrogel subunits that allows the passage ofsuch biological macromolecules.

In some embodiments, hydrogel formation on a substrate occurs before,contemporaneously with, or after features (e.g., beads) are attached tothe substrate. For example, when a capture probe is attached (e.g.,directly or indirectly) to a substrate, hydrogel formation can beperformed on the substrate already containing the capture probes.

(d) Arrays

In many of the methods described herein, features (as described furtherbelow) are collectively positioned on a substrate. An “array” is aspecific arrangement of a plurality of features that is either irregularor forms a regular pattern. Individual features in the array differ fromone another based on their relative spatial locations. In general, atleast two of the plurality of features in the array include a distinctcapture probe (e.g., any of the examples of capture probes describedherein).

Arrays can be used to measure large numbers of analytes simultaneously.In some embodiments, oligonucleotides are used, at least in part, tocreate an array. For example, one or more copies of a single species ofoligonucleotide (e.g., capture probe) can correspond to or be directlyor indirectly attached to a given feature in the array. In someembodiments, a given feature in the array includes two or more speciesof oligonucleotides (e.g., capture probes). In some embodiments, the twoor more species of oligonucleotides (e.g., capture probes) attacheddirectly or indirectly to a given feature on the array include a common(e.g., identical) spatial barcode.

(i) Arrays for Analyte Capture

In some embodiments, an array can include a capture probe attacheddirectly or indirectly to the substrate. The capture probe can include acapture domain (e.g., a nucleotide sequence) that can specifically bind(e.g., hybridize) to a target analyte (e.g., mRNA, DNA, or protein)within a sample. In some embodiments, the binding of the capture probeto the target (e.g., hybridization) can be detected and quantified bydetection of a visual signal, e.g., a fluorophore, a heavy metal (e.g.,silver ion), or chemiluminescent label, which has been incorporated intothe target. In some embodiments, the intensity of the visual signalcorrelates with the relative abundance of each analyte in the biologicalsample. Since an array can contain thousands or millions of captureprobes (or more), an array can interrogate many analytes in parallel.

In some embodiments, a substrate includes one or more capture probesthat are designed to capture analytes from one or more organisms. In anon-limiting example, a substrate can contain one or more capture probesdesigned to capture mRNA from one organism (e.g., a human) and one ormore capture probes designed to capture DNA from a second organism(e.g., a bacterium).

The capture probes can be attached to a substrate or feature using avariety of techniques. In some embodiments, the capture probe isdirectly attached to a feature that is fixed on an array. In someembodiments, the capture probes are immobilized to a substrate bychemical immobilization. For example, a chemical immobilization can takeplace between functional groups on the substrate and correspondingfunctional elements on the capture probes. Exemplary correspondingfunctional elements in the capture probes can either be an inherentchemical group of the capture probe, e.g., a hydroxyl group, or afunctional element can be introduced on to the capture probe. An exampleof a functional group on the substrate is an amine group. In someembodiments, the capture probe to be immobilized includes a functionalamine group or is chemically modified in order to include a functionalamine group. Means and methods for such a chemical modification are wellknown in the art.

In some embodiments, the capture probe is a nucleic acid. In someembodiments, the capture probe is immobilized on a substrate or featurevia its 5′ end. In some embodiments, the capture probe is immobilized ona substrate or feature via its 5′ end and includes from the 5′ to 3′end: one or more barcodes (e.g., a spatial barcode and/or a UMI) and oneor more capture domains. In some embodiments, the capture probe isimmobilized on a substrate or feature via its 5′ end and includes fromthe 5′ to 3′ end: one barcode (e.g., a spatial barcode or a UMI) and onecapture domain. In some embodiments, the capture probe is immobilized ona substrate or feature via its 5′ end and includes from the 5′ to 3′end: a cleavage domain, a functional domain, one or more barcodes (e.g.,a spatial barcode and/or a UMI), and a capture domain.

In some embodiments, the capture probe is immobilized on a substrate orfeature via its 5′ end and includes from the 5′ to 3′ end: a cleavagedomain, a functional domain, one or more barcodes (e.g., a spatialbarcode and/or a UMI), a second functional domain, and a capture domain.In some embodiments, the capture probe is immobilized on a substrate orfeature via its 5′ end and includes from the 5′ to 3′ end: a cleavagedomain, a functional domain, a spatial barcode, a UMI, and a capturedomain. In some embodiments, the capture probe is immobilized on asubstrate or feature via its 5′ end and does not include a spatialbarcode. In some embodiments, the capture probe is immobilized on asubstrate or feature via its 5′ end and does not include a UMI. In someembodiments, the capture probe includes a sequence for initiating asequencing reaction.

In some embodiments, the capture probe is immobilized on a substrate orfeature via its 3′ end. In some embodiments, the capture probe isimmobilized on a substrate or feature via its 3′ end and includes fromthe 3′ to 5′ end: one or more barcodes (e.g., a spatial barcode and/or aUMI) and one or more capture domains. In some embodiments, the captureprobe is immobilized on a substrate or feature via its 3′ end andincludes from the 3′ to 5′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probe isimmobilized on a substrate or feature via its 3′ end and includes fromthe 3′ to 5′ end: a cleavage domain, a functional domain, one or morebarcodes (e.g., a spatial barcode and/or a UMI), and a capture domain.In some embodiments, the capture probe is immobilized on a substrate orfeature via its 3′ end and includes from the 3′ to 5′ end: a cleavagedomain, a functional domain, a spatial barcode, a UMI, and a capturedomain.

The localization of the functional group within the capture probe to beimmobilized can be used to control and shape the binding behavior and/ororientation of the capture probe, e.g., the functional group can beplaced at the 5′ or 3′ end of the capture probe or within the sequenceof the capture probe. In some embodiments, a capture probe can furtherinclude a substrate. A typical substrate for a capture probe to beimmobilized includes moieties which are capable of binding to suchcapture probes, e.g., to amine-functionalized nucleic acids. Examples ofsuch substrates are carboxy, aldehyde, or epoxy substrates.

In some embodiments, the substrates on which capture probes can beimmobilized can be chemically activated, e.g., by the activation offunctional groups available on the substrate. The term “activatedsubstrate” relates to a material in which interacting or reactivechemical functional groups are established or enabled by chemicalmodification procedures. For example, a substrate including carboxylgroups can be activated before use. Furthermore, certain substratescontain functional groups that can react with specific moieties alreadypresent in the capture probes.

In some embodiments, a covalent linkage is used to directly couple acapture probe to a substrate. In some embodiments a capture probe isindirectly coupled to a substrate through a linker separating the“first” nucleotide of the capture probe from the substrate, e.g., achemical linker. In some embodiments, a capture probe does not binddirectly to the substrate, but interacts indirectly, for example bybinding to a molecule which itself binds directly or indirectly to thesubstrate. In some embodiments, the capture probe is indirectly attachedto a substrate (e.g., attached to a substrate via a solution including apolymer).

In some embodiments where the capture probe is immobilized on a featureof the array indirectly, e.g., via hybridization to a surface probecapable of binding the capture probe, the capture probe can furtherinclude an upstream sequence (5′ to the sequence that hybridizes to thenucleic acid, e.g., RNA of the tissue sample) that is capable ofhybridizing to 5′ end of a surface probe. Alone, the capture domain ofthe capture probe can be seen as a capture domain oligonucleotide, whichcan be used in the synthesis of the capture probe in embodiments wherethe capture probe is immobilized on the array indirectly.

In some embodiments, a substrate is comprised of an inert material ormatrix (e.g., glass slides) that has been functionalized by, forexample, treating the substrate with a material comprising reactivegroups which enable immobilization of capture probes. See, for example,WO 2017/019456, the entire contents of which is herein incorporated byreference. Non-limiting examples include polyacrylamide hydrogelssupported on an inert substrate (e.g., glass slide; see WO 2005/065814and U.S. Patent Application No. 2008/0280773, the entire contents ofwhich is incorporated herein by reference).

In some embodiments, functionalized biomolecules (e.g., capture probes)are immobilized on a functionalized substrate using covalent methods.Methods for covalent attachment include, for example, condensation ofamines and activated carboxylic esters (e.g., N-hydroxysuccinimideesters); condensation of amine and aldehydes under reductive aminationconditions; and cycloaddition reactions such as the Diels-Alder [4+2]reaction, 1,3-dipolar cycloaddition reactions, and [2+2] cycloadditionreactions. Methods for covalent attachment also include, for example,click chemistry reactions, including [3+2] cycloaddition reactions(e.g., Huisgen 1,3-dipolar cycloaddition reaction andcopper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)); thiol-enereactions; the Diels-Alder reaction and inverse electron demandDiels-Alder reaction; [4+1] cycloaddition of isonitriles and tetrazines;and nucleophilic ring-opening of small carbocycles (e.g., epoxideopening with amino oligonucleotides). Methods for covalent attachmentalso include, for example, maleimides and thiols; and para-nitrophenylester-functionalized oligonucleotides and polylysine-functionalizedsubstrate. Methods for covalent attachment also include, for example,disulfide reactions; radical reactions (see, e.g., U.S. Pat. No.5,919,626, the entire contents of which are herein incorporated byreference); and hydrazide-functionalized substrate (e.g., wherein thehydrazide functional group is directly or indirectly attached to thesubstrate) and aldehyde-functionalized oligonucleotides (see, e.g.,Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918, theentire contents of which are herein incorporated by reference).

In some embodiments, functionalized biomolecules (e.g., capture probes)are immobilized on a functionalized substrate using photochemicalcovalent methods. Methods for photochemical covalent attachment include,for example, immobilization of antraquinone-conjugated oligonucleotides(see, e.g., Koch et al. (2000) Bioconjugate Chem. 11, 474-483, theentire contents of which is herein incorporated by reference).

In some embodiments, functionalized biomolecules (e.g., capture probes)are immobilized on a functionalized substrate using non-covalentmethods. Methods for non-covalent attachment include, for example,biotin-functionalized oligonucleotides and streptavidin-treatedsubstrates (see, e.g., Holmstrom et al. (1993) Analytical Biochemistry209, 278-283 and Gilles et al. (1999) Nature Biotechnology 17, 365-370,the entire contents of which are herein incorporated by reference).

In some embodiments, an oligonucleotide (e.g., a capture probe) can beattached to a substrate or feature according to the methods set forth inU.S. Pat. Nos. 6,737,236, 7,259,258, 7,375,234, 7,427,678, 5,610,287,5,807,522, 5,837,860, and 5,472,881; U.S. Patent Application PublicationNos. 2008/0280773 and 2011/0059865; Shalon et al. (1996) GenomeResearch, 639-645; Rogers et al. (1999) Analytical Biochemistry 266,23-30; Stimpson et al. (1995) Proc. Natl. Acad. Sci. USA 92, 6379-6383;Beattie et al. (1995) Clin. Chem. 45, 700-706; Lamture et al. (1994)Nucleic Acids Research 22, 2121-2125; Beier et al. (1999) Nucleic AcidsResearch 27, 1970-1977; Joos et al. (1997)Analytical Biochemistry 247,96-101; Nikiforov et al. (1995) Analytical Biochemistry 227, 201-209;Timofeev et al. (1996) Nucleic Acids Research 24, 3142-3148; Chrisey etal. (1996) Nucleic Acids Research 24, 3031-3039; Guo et al. (1994)Nucleic Acids Research 22, 5456-5465; Running and Urdea (1990)BioTechniques 8, 276-279; Fahy et al. (1993) Nucleic Acids Research 21,1819-1826; Zhang et al. (1991) 19, 3929-3933; and Rogers et al. (1997)Gene Therapy 4, 1387-1392. The entire contents of each of the foregoingdocuments is incorporated herein by reference.

(ii) Features

A “feature” is an entity that acts as a support or repository forvarious molecular entities used in sample analysis. In some embodiments,some or all of the features in an array are functionalized for analytecapture. In some embodiments, functionalized features include one ormore capture probe(s). Examples of features include, but are not limitedto, a bead, a spot of any two- or three-dimensional geometry (e.g., anink jet spot, a masked spot, a square on a grid), a well, and a hydrogelpad. In some embodiments, features are directly or indirectly attachedor fixed to a substrate. In some embodiments, the features are notdirectly or indirectly attached or fixed to a substrate, but instead,for example, are disposed within an enclosed or partially enclosed threedimensional space (e.g., wells or divots).

In addition to those above, a wide variety of other features can be usedto form the arrays described herein. For example, in some embodiments,features that are formed from polymers and/or biopolymers that are jetprinted, screen printed, or electrostatically deposited on a substratecan be used to form arrays. Jet printing of biopolymers is described,for example, in PCT Patent Application Publication No. WO 2014/085725.Jet printing of polymers is described, for example, in de Gans et al.,Adv Mater. 16(3): 203-213 (2004). Methods for electrostatic depositionof polymers and biopolymers are described, for example, in Hoyer et al.,Anal. Chem. 68(21): 3840-3844 (1996). The entire contents of each of theforegoing references are incorporated herein by reference.

As another example, in some embodiments, features are formed by metallicmicro- or nanoparticles. Suitable methods for depositing such particlesto form arrays are described, for example, in Lee et al., Beilstein J.Nanotechnol. 8: 1049-1055 (2017), the entire contents of which areincorporated herein by reference.

As a further example, in some embodiments, features are formed bymagnetic particles that are assembled on a substrate. Examples of suchparticles and methods for assembling arrays are described in Ye et al.,Scientific Reports 6: 23145 (2016), the entire contents of which areincorporated herein by reference.

As another example, in some embodiments, features correspond to regionsof a substrate in which one or more optical labels have beenincorporated, and/or which have been altered by a process such aspermanent photobleaching. Suitable substrates to implement features inthis manner include a wide variety of polymers, for example. Methods forforming such features are described, for example, in Moshrefzadeh etal., Appl. Phys. Lett. 62: 16 (1993), the entire contents of which areincorporated herein by reference.

As yet another example, in some embodiments, features can correspond tocolloidal particles assembled (e.g., via self-assembly) to form anarray. Suitable colloidal particles are described for example in Sharma,Resonance 23(3): 263-275 (2018), the entire contents of which areincorporated herein by reference.

As a further example, in some embodiments, features can be formed viaspot-array photopolymerization of a monomer solution on a substrate. Inparticular, two-photon and three-photon polymerization can be used tofabricate features of relatively small (e.g., sub-micron) dimensions.Suitable methods for preparing features on a substrate in this mannerare described for example in Nguyen et al., Materials Today 20(6):314-322 (2017), the entire contents of which are incorporated herein byreference.

In some embodiments, features are directly or indirectly attached orfixed to a substrate that is liquid permeable. In some embodiments,features are directly or indirectly attached or fixed to a substratethat is biocompatible. In some embodiments, features are directly orindirectly attached or fixed to a substrate that is a hydrogel.

FIG. 12 depicts an exemplary arrangement of barcoded features within anarray. From left to right, FIG. 12 shows (L) a slide including sixspatially-barcoded arrays, (C) an enlarged schematic of one of the sixspatially-barcoded arrays, showing a grid of barcoded features inrelation to a biological sample, and (R) an enlarged schematic of onesection of an array, showing the specific identification of multiplefeatures within the array (labelled as ID578, ID579, ID560, etc.).

(1) Beads

A “bead” can be a particle. A bead can be porous, non-porous, solid,semi-solid, and/or a combination thereof. In some embodiments, a beadcan be dissolvable, disruptable, and/or degradable, whereas in certainembodiments, a bead is not degradable. A semi-solid bead can be aliposomal bead. Solid beads can include metals including, withoutlimitation, iron oxide, gold, and silver. In some embodiments, the beadcan be a silica bead. In some embodiments, the bead can be rigid. Insome embodiments, the bead can be flexible and/or compressible.

The bead can be a macromolecule. The bead can be formed of nucleic acidmolecules bound together. The bead can be formed via covalent ornon-covalent assembly of molecules (e.g., macromolecules), such asmonomers or polymers. Polymers or monomers can be natural or synthetic.Polymers or monomers can be or include, for example, nucleic acidmolecules (e.g., DNA or RNA).

A bead can be rigid, or flexible and/or compressible. A bead can includea coating including one or more polymers. Such a coating can bedisruptable or dissolvable. In some embodiments, a bead includes aspectral or optical label (e.g., dye) attached directly or indirectly(e.g., through a linker) to the bead. For example, a bead can beprepared as a colored preparation (e.g., a bead exhibiting a distinctcolor within the visible spectrum) that can change color (e.g.,colorimetric beads) upon application of a desired stimulus (e.g., heatand/or chemical reaction) to form differently colored beads (e.g.,opaque and/or clear beads).

A bead can include natural and/or synthetic materials. For example, abead can include a natural polymer, a synthetic polymer or both naturaland synthetic polymers.

In some embodiments, a bead is a degradable bead. A degradable bead caninclude one or more species (e.g., disulfide linkers, primers, otheroligonucleotides, etc.) with a labile bond such that, when thebead/species is exposed to the appropriate stimuli, the labile bond isbroken and the bead degrades. The labile bond can be a chemical bond(e.g., covalent bond, ionic bond) or can be another type of physicalinteraction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some embodiments, a cross-linker used togenerate a bead can include a labile bond. Upon exposure to theappropriate conditions, the labile bond can be broken and the beaddegraded. For example, upon exposure of a polyacrylamide gel beadincluding cystamine cross-linkers to a reducing agent, the disulfidebonds of the cystamine can be broken and the bead degraded.

Degradation can refer to the disassociation of a bound or entrainedspecies (e.g., disulfide linkers, primers, other oligonucleotides, etc.)from a bead, both with and without structurally degrading the physicalbead itself. For example, entrained species can be released from beadsthrough osmotic pressure differences due to, for example, changingchemical environments. By way of example, alteration of bead porevolumes due to osmotic pressure differences can generally occur withoutstructural degradation of the bead itself. In some embodiments, anincrease in pore volume due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In some embodiments,osmotic shrinking of a bead can cause a bead to better retain anentrained species due to pore volume contraction.

Any suitable agent that can degrade beads can be used. In someembodiments, changes in temperature or pH can be used to degradethermo-sensitive or pH-sensitive bonds within beads. In someembodiments, chemical degrading agents can be used to degrade chemicalbonds within beads by oxidation, reduction or other chemical changes.For example, a chemical degrading agent can be a reducing agent, such asDTT, where DTT can degrade the disulfide bonds formed between across-linker and gel precursors, thus degrading the bead. In someembodiments, a reducing agent can be added to degrade the bead, whichcan cause the bead to release its contents. Examples of reducing agentscan include, without limitation, dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof.

Any of a variety of chemical agents can be used to trigger thedegradation of beads. Examples of chemical agents include, but are notlimited to, pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead can be formed from materials that includedegradable chemical cross-linkers, such as N,N′-bis-(acryloyl)cystamine(BAC) or cystamine. Degradation of such degradable cross-linkers can beaccomplished through any variety of mechanisms. In some examples, a beadcan be contacted with a chemical degrading agent that can induceoxidation, reduction or other chemical changes. For example, a chemicaldegrading agent can be a reducing agent, such as dithiothreitol (DTT).Additional examples of reducing agents can include β-mercaptoethanol,(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA),tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.

In some embodiments, exposure to an aqueous solution, such as water, cantrigger hydrolytic degradation, and thus degradation of the bead. Beadscan also be induced to release their contents upon the application of athermal stimulus. A change in temperature can cause a variety of changesto a bead. For example, heat can cause a solid bead to liquefy. A changein heat can cause melting of a bead such that a portion of the beaddegrades. In some embodiments, heat can increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat canalso act upon heat-sensitive polymers used as materials to constructbeads.

Where degradable beads are used, it can be beneficial to avoid exposingsuch beads to the stimulus or stimuli that cause such degradation priorto a given time, in order to, for example, avoid premature beaddegradation and issues that arise from such degradation, including forexample poor flow characteristics and aggregation. By way of example,where beads include reducible cross-linking groups, such as disulfidegroups, it will be desirable to avoid contacting such beads withreducing agents, e.g., DTT or other disulfide cleaving reagents. In suchembodiments, treatment of the beads described herein will, in someembodiments be provided free of reducing agents, such as DTT. Becausereducing agents are often provided in commercial enzyme preparations, itcan be desirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that can be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations refer to a preparation having less than about 1/10th, lessthan about 1/50th, or less than about 1/100th of the lower ranges forsuch materials used in degrading the beads. For example, for DTT, thereducing agent free preparation can have less than about 0.01 millimolar(mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or less than about 0.0001mM DTT. In some embodiments, the amount of DTT can be undetectable.

A degradable bead can be useful to more quickly release an attachedcapture probe (e.g., a nucleic acid molecule, a spatial barcodesequence, and/or a primer) from the bead when the appropriate stimulusis applied to the bead as compared to a bead that does not degrade. Forexample, for a species bound to an inner surface of a porous bead or inthe case of an encapsulated species, the species can have greatermobility and accessibility to other species in solution upon degradationof the bead. In some embodiments, a species can also be attached to adegradable bead via a degradable linker (e.g., disulfide linker). Thedegradable linker can respond to the same stimuli as the degradable beador the two degradable species can respond to different stimuli. Forexample, a capture probe having one or more spatial barcodes can beattached, via a disulfide bond, to a polyacrylamide bead includingcystamine. Upon exposure of the spatially-barcoded bead to a reducingagent, the bead degrades and the capture probe having the one or morespatial barcode sequences is released upon breakage of both thedisulfide linkage between the capture probe and the bead and thedisulfide linkages of the cystamine in the bead.

The addition of multiple types of labile bonds to a bead can result inthe generation of a bead capable of responding to varied stimuli. Eachtype of labile bond can be sensitive to an associated stimulus (e.g.,chemical stimulus, light, temperature, pH, enzymes, etc.) such thatrelease of reagents attached to a bead via each labile bond can becontrolled by the application of the appropriate stimulus. Somenon-limiting examples of labile bonds that can be coupled to a precursoror bead include an ester linkage (e.g., cleavable with an acid, a base,or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodiumperiodate), a Diels-Alder linkage (e.g., cleavable via heat), a sulfonelinkage (e.g., cleavable via a base), a silyl ether linkage (e.g.,cleavable via an acid), a glycosidic linkage (e.g., cleavable via anamylase), a peptide linkage (e.g., cleavable via a protease), or aphosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)).A bond can be cleavable via other nucleic acid molecule targetingenzymes, such as restriction enzymes (e.g., restriction endonucleases).Such functionality can be useful in controlled release of reagents froma bead. In some embodiments, another reagent including a labile bond canbe linked to a bead after gel bead formation via, for example, anactivated functional group of the bead as described above. In someembodiments, a gel bead including a labile bond is reversible. In someembodiments, a gel bead with a reversible labile bond is used to captureone or more regions of interest of a biological sample. For example,without limitation, a bead including a thermolabile bond can be heatedby a light source (e.g., a laser) that causes a change in the gel beadthat facilitates capture of a biological sample in contact with the gelbead. Capture probes having one or more spatial barcodes that arereleasably, cleavably, or reversibly attached to the beads describedherein include capture probes that are released or releasable throughcleavage of a linkage between the capture probe and the bead, or thatare released through degradation of the underlying bead itself, allowingthe capture probes having the one or more spatial barcodes to beaccessed or become accessible by other reagents, or both.

Beads can have different physical properties. Physical properties ofbeads can be used to characterize the beads. Non-limiting examples ofphysical properties of beads that can differ include volume, shape,circularity, density, symmetry, and hardness. For example, beads can beof different volumes. Beads of different diameters can be obtained byusing microfluidic channel networks configured to provide beads of aspecific volume (e.g., based on channel sizes, flow rates, etc.). Insome embodiments, beads have different hardness values that can beobtained by varying the concentration of polymer used to generate thebeads. In some embodiments, a spatial barcode attached to a bead can bemade optically detectable using a physical property of the captureprobe. For example, a nucleic acid origami, such as a deoxyribonucleicacid (DNA) origami, can be used to generate an optically detectablespatial barcode. To do so, a nucleic acid molecule, or a plurality ofnucleic acid molecules, can be folded to create two- and/orthree-dimensional geometric shapes. The different geometric shapes canbe optically detected.

In some embodiments, special types of nanoparticles with more than onedistinct physical property can be used to make the beads physicallydistinguishable. For example, Janus particles with both hydrophilic andhydrophobic surfaces can be used to provide unique physical properties.

A bead can generally be of any suitable shape. Examples of bead shapesinclude, but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, cuboidal, hexagonal, and variationsthereof. In some embodiments, non-spherical (e.g., hexagonal, cuboidal,shaped beads can assemble more closely (e.g., tighter) than sphericalshaped beads. In some embodiments, beads can self-assemble into amonolayer. A cross section (e.g., a first cross-section) can correspondto a diameter or maximum cross-sectional dimension of the bead. In someembodiments, the bead can be approximately spherical. In suchembodiments, the first cross-section can correspond to the diameter ofthe bead. In some embodiments, the bead can be approximatelycylindrical. In such embodiments, the first cross-section can correspondto a diameter, length, or width along the approximately cylindricalbead.

Beads can be of uniform size or heterogeneous size. “Polydispersity”generally refers to heterogeneity of sizes of molecules or particles.The polydispersity index (PDI) of a bead can be calculated using theequation PDI=Mw/Mn, where Mw is the weight-average molar mass and Mn isthe number-average molar mass. In certain embodiments, beads can beprovided as a population or plurality of beads having a relativelymonodisperse size distribution. Where it can be desirable to providerelatively consistent amounts of reagents, maintaining relativelyconsistent bead characteristics, such as size, can contribute to theoverall consistency.

The bead can include one or more cross-sections that can be the same ordifferent. In some embodiments, the bead can have a first cross-sectionthat is different from a second cross-section.

In some embodiments, a bead is able to identify multiple analytes (e.g.,nucleic acids, proteins, chromatin, metabolites, drugs, gRNA, andlipids) from a single cell. In some embodiments, a bead is able toidentify a single analyte from a single cell (e.g., mRNA).

A bead can have a tunable pore volume. The pore volume can be chosen to,for instance, retain denatured nucleic acids. The pore volume can bechosen to maintain diffusive permeability to exogenous chemicals such assodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors.A bead can be formed of a biocompatible and/or biochemically compatiblematerial, and/or a material that maintains or enhances cell viability. Abead can be formed from a material that can be depolymerized thermally,chemically, enzymatically, and/or optically.

In some embodiments, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. Swelling of the beads can be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field.

The swelling of the beads can be accomplished by various swellingmethods. In some embodiments, swelling is reversible (e.g., bysubjecting beads to conditions that promote de-swelling). In someembodiments, the de-swelling of the beads is accomplished, for instance,by transferring the beads in a thermodynamically unfavorable solvent,subjecting the beads to lower or higher temperatures, subjecting thebeads to a lower or higher ion concentration, and/or adding or removingan electric field. The de-swelling of the beads can be accomplished byvarious de-swelling methods. In some embodiments, de-swelling isreversible (e.g., subject beads to conditions that promote swelling). Insome embodiments, the de-swelling of beads can include transferring thebeads to cause pores in the bead to shrink. The shrinking can thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance created can be due to steric interactionsbetween the reagents and the interiors of the beads. The transfer can beaccomplished microfluidically. For instance, the transfer can beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore volumeof the beads can be adjusted by changing the polymer composition of thebead.

A bead can include a polymer that is responsive to temperature so thatwhen the bead is heated or cooled, the characteristics or dimensions ofthe bead can change. For example, a polymer can includepoly(N-isopropylacrylamide). A gel bead can includepoly(N-isopropylacrylamide) and when heated the gel bead can decrease inone or more dimensions (e.g., a cross-sectional diameter, multiplecross-sectional diameters). A temperature sufficient for changing one ormore characteristics of the gel bead can be, for example, at least about0 degrees Celsius (° C.), 1° C., 2° C., 3° C., 4° C., 5° C., 10° C., orhigher. For example, the temperature can be about 4° C. In someembodiments, a temperature sufficient for changing one or morecharacteristics of the gel bead can be, for example, at least about 25°C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., or higher. Forexample, the temperature can be about 37° C.

Functionalization of beads for attachment of capture probes can beachieved through a wide range of different approaches, including,without limitation, activation of chemical groups within a polymer,incorporation of active or activatable functional groups in the polymerstructure, or attachment at the pre-polymer or monomer stage in beadproduction. The bead can be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids, metabolites,peptides, or other analytes.

In some embodiments, a bead can contain molecular precursors (e.g.,monomers or polymers), which can form a polymer network viapolymerization of the molecular precursors. In some embodiments, aprecursor can be an already polymerized species capable of undergoingfurther polymerization via, for example, a chemical cross-linkage. Insome embodiments, a precursor can include one or more of an acrylamideor a methacrylamide monomer, oligomer, or polymer. In some embodiments,the bead can include prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads can be prepared usingprepolymers. In some embodiments, a bead can contain individual polymersthat can be further polymerized together (e.g., to form a co-polymer).In some embodiments, a bead can be generated via polymerization ofdifferent precursors, such that they include mixed polymers,co-polymers, and/or block co-polymers. In some embodiments, a bead caninclude covalent or ionic bonds between polymeric precursors (e.g.,monomers, oligomers, and linear polymers), nucleic acid molecules (e.g.,oligonucleotides), primers, and other entities. In some embodiments,covalent bonds can be carbon-carbon bonds or thioether bonds.

Cross-linking of polymers can be permanent or reversible, depending uponthe particular cross-linker used. Reversible cross-linking can allow thepolymer to linearize or dissociate under appropriate conditions. In someembodiments, reversible cross-linking can also allow for reversibleattachment of a material bound to the surface of a bead. In someembodiments, a cross-linker can form a disulfide linkage. In someembodiments, a chemical cross-linker forming a disulfide linkage can becystamine or a modified cystamine.

For example, where the polymer precursor material includes a linearpolymer material, such as a linear polyacrylamide, PEG, or other linearpolymeric material, the activation agent can include a cross-linkingagent, or a chemical that activates a cross-linking agent within formeddroplets. Likewise, for polymer precursors that include polymerizablemonomers, the activation agent can include a polymerization initiator.For example, in certain embodiments, where the polymer precursorincludes a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) can be provided, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or other conditions sufficient to polymerize or gel theprecursors. The conditions sufficient to polymerize or gel theprecursors can include exposure to heating, cooling, electromagneticradiation, and/or light.

Following polymerization or gelling, a polymer or gel can be formed. Thepolymer or gel can be diffusively permeable to chemical or biochemicalreagents. The polymer or gel can be diffusively impermeable tomacromolecular constituents. The polymer or gel can include one or moreof disulfide cross-linked polyacrylamide, agarose, alginate, polyvinylalcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol,PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid,collagen, fibrin, gelatin, or elastin. The polymer or gel can includeany other polymer or gel.

In some embodiments, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides, capture probes). Cystamine (including modifiedcystamines), for example, is an organic agent including a disulfide bondthat can be used as a cross-linker agent between individual monomeric orpolymeric precursors of a bead. Polyacrylamide can be polymerized in thepresence of cystamine or a species including cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads including disulfidelinkages (e.g., chemically degradable beads includingchemically-reducible cross-linkers). The disulfide linkages can permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some embodiments, chitosan, a linear polysaccharide polymer, can becross-linked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers can be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some embodiments, a bead can include an acrydite moiety, which incertain aspects can be used to attach one or more capture probes to thebead. In some embodiments, an acrydite moiety can refer to an acryditeanalogue generated from the reaction of acrydite with one or morespecies (e.g., disulfide linkers, primers, other oligonucleotides,etc.), such as, without limitation, the reaction of acrydite with othermonomers and cross-linkers during a polymerization reaction. Acryditemoieties can be modified to form chemical bonds with a species to beattached, such as a capture probe. Acrydite moieties can be modifiedwith thiol groups capable of forming a disulfide bond or can be modifiedwith groups already including a disulfide bond. The thiol or disulfide(via disulfide exchange) can be used as an anchor point for a species tobe attached or another part of the acrydite moiety can be used forattachment. In some embodiments, attachment can be reversible, such thatwhen the disulfide bond is broken (e.g., in the presence of a reducingagent), the attached species is released from the bead. In someembodiments, an acrydite moiety can include a reactive hydroxyl groupthat can be used for attachment of species.

In some embodiments, precursors (e.g., monomers or cross-linkers) thatare polymerized to form a bead can include acrydite moieties, such thatwhen a bead is generated, the bead also includes acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g., anoligonucleotide), which can include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or one or more capture probes. The one or morecapture probes can include sequences that are the same for all captureprobes coupled to a given bead and/or sequences that are differentacross all capture probes coupled to the given bead. The capture probecan be incorporated into the bead. In some embodiments, the captureprobe can be incorporated or attached to the bead such that the captureprobe retains a free 3′ end. In some embodiments, the capture probe canbe incorporated or attached to the bead such that the capture proberetains a free 5′ end. In some embodiments, beads can be functionalizedsuch that each bead contains a plurality of different capture probes.For example, a bead can include a plurality of capture probes e.g.,Capture Probe 1, Capture Probe 2, and Capture Probe 3, and each ofCapture Probes 1, Capture Probes 2, and Capture Probes 3 contain adistinct capture domain (e.g., capture domain of Capture Probe 1includes a poly(dT) capture domain, capture domain of Capture Probe 2includes a gene-specific capture domain, and capture domain of CaptureProbe 3 includes a CRISPR-specific capture domain). By functionalizingbeads to contain a plurality of different capture domains per bead, thelevel of multiplex capability for analyte detection can be improved.

In some embodiments, precursors (e.g., monomers or cross-linkers) thatare polymerized to form a bead can include a functional group that isreactive or capable of being activated such that when it becomesreactive it can be polymerized with other precursors to generate beadsincluding the activated or activatable functional group. The functionalgroup can then be used to attach additional species (e.g., disulfidelinkers, primers, other oligonucleotides, etc.) to the beads.

Beads including disulfide linkages in their polymeric network can befunctionalized with additional species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages can be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies including another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some embodiments, free thiols of the beadscan react with any other suitable group. For example, free thiols of thebeads can react with species including an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species including the acrydite islinked to the bead. In some embodiments, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlcan be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In someembodiments, a low concentration of reducing agent (e.g., molecules ofreducing agent:gel bead ratios) of less than or equal to about1:100,000,000,000, less than or equal to about 1:10,000,000,000, lessthan or equal to about 1:1,000,000,000, less than or equal to about1:100,000,000, less than or equal to about 1:10,000,000, less than orequal to about 1:1,000,000, less than or equal to about 1:100,000, orless than or equal to about 1:10,000) can be used for reduction.Controlling the number of disulfide linkages that are reduced to freethiols can be useful in ensuring bead structural integrity duringfunctionalization. In some embodiments, optically-active agents, such asfluorescent dyes can be coupled to beads via free thiol groups of thebeads and used to quantify the number of free thiols present in a beadand/or track a bead.

In some embodiments, addition of moieties to a bead after bead formationcan be advantageous. For example, addition of a capture probe after beadformation can avoid loss of the species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) during chain transfer terminationthat can occur during polymerization. In some embodiments, smallerprecursors (e.g., monomers or cross linkers that do not include sidechain groups and linked moieties) can be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome embodiments, functionalization after bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some embodiments, the generated hydrogel can possess anupper critical solution temperature (UCST) that can permit temperaturedriven swelling and collapse of a bead. Such functionality can aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization can also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading can also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

Reagents can be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such reagents can or cannotparticipate in polymerization. Such reagents can be entered intopolymerization reaction mixtures such that generated beads include thereagents upon bead formation. In some embodiments, such reagents can beadded to the beads after formation. Such reagents can include, forexample, capture probes (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,chemical substrates, buffers), reagents for nucleic acid modificationreactions such as polymerization, ligation, or digestion, and/orreagents for template preparation (e.g., tagmentation) for one or moresequencing platforms (e.g., Nextera® for Illumina®). Such reagents caninclude one or more enzymes described herein, including withoutlimitation, polymerase, reverse transcriptase, restriction enzymes(e.g., endonuclease), transposase, ligase, proteinase K, DNAse, etc.Such reagents can also or alternatively include one or more reagentssuch as lysis agents, inhibitors, inactivating agents, chelating agents,stimulus agents. Trapping of such reagents can be controlled by thepolymer network density generated during polymerization of precursors,control of ionic charge within the bead (e.g., via ionic species linkedto polymerized species), or by the release of other species.Encapsulated reagents can be released from a bead upon bead degradationand/or by application of a stimulus capable of releasing the reagentsfrom the bead. In some embodiments, the beads or bead arrangements canbe incubated in permeabilization reagents as described herein.

In some embodiments, the beads can also include (e.g., encapsulate orhave attached thereto) a plurality of capture probes that includespatial barcodes, and the optical properties of the spatial barcodes canbe used for optical detection of the beads. For example, the absorbanceof light by the spatial barcodes can be used to distinguish the beadsfrom one another. In some embodiments, a detectable label can directlyor indirectly attach to a spatial barcode and provide optical detectionof the bead. In some embodiments, each bead in a group of one or morebeads has a unique detectable label, and detection of the uniquedetectable label determines the location of the spatial barcode sequenceassociated with the bead.

Optical properties giving rise to optical detection of beads can be dueto optical properties of the bead surface (e.g., a detectable labelattached to the bead), or optical properties from the bulk region of thebead (e.g., a detectable label incorporated during bead formation or anoptical property of the bead itself). In some embodiments, a detectablelabel can be associated with a bead or one or more moieties coupled tothe bead.

In some embodiments, the beads include a plurality of detectable labels.For example, a fluorescent dye can be attached to the surface of thebeads and/or can be incorporated into the beads. Different intensitiesof the different fluorescent dyes can be used to increase the number ofoptical combinations that can be used to differentiate between beads.For example, if N is the number of fluorescent dyes (e.g., between 2 and10 fluorescent dyes, such as 4 fluorescent dyes) and M is the possibleintensities for the dyes (e.g., between 2 and 50 intensities, such as 20intensities), then M^(N) are the possible distinct optical combinations.In one example, 4 fluorescent dyes with 20 possible intensities can beused to generate 160,000 distinct optical combinations.

One or more optical properties of the beads or biological contents, suchas cells or nuclei, can be used to distinguish the individual beads orbiological contents from other beads or biological contents. In someembodiments, the beads are made optically detectable by including adetectable label having optical properties to distinguish the beads fromone another.

In some embodiments, optical properties of the beads can be used foroptical detection of the beads. For example, without limitation, opticalproperties can include absorbance, birefringence, color, fluorescence,luminosity, photosensitivity, reflectivity, refractive index,scattering, or transmittance. For example, beads can have differentbirefringence values based on degree of polymerization, chain length, ormonomer chemistry.

In some embodiments, nanobeads, such as quantum dots or Janus beads, canbe used as optical labels or components thereof. For example, a quantumdot can be attached to a spatial barcode of a bead.

Optical labels of beads can provide enhanced spectral resolution todistinguish (e.g., identify) between beads with unique spatial barcodes(e.g., beads including unique spatial barcode sequences). That is, thebeads are manufactured in a way that the optical labels and the barcodeson the beads (e.g., spatial barcodes) are correlated with each other. Insome aspects, the beads can be loaded into a flowcell such that beadsare arrayed in a closely packed manner (e.g., single-cell resolution).Imaging can be performed, and the spatial location of the barcodes canbe determined (e.g., based on information from a look-up table (LUT)).The optical labels for spatial profiling allow for quick deconvolutionof bead-barcode (e.g., spatial barcode) identify.

In some examples, a lookup table (LUT) can be used to associate aproperty (e.g., an optical label, such as a color and/or intensity) ofthe bead with the barcode sequence. The property may derive from theparticle (e.g., bead) or an optical label associated with the bead. Thebeads can be imaged to obtain optical information of the bead,including, for example, the property (e.g., color and/or intensity) ofthe bead or the optical label associated with the bead, and opticalinformation of the biological sample. For example, an image can includeoptical information in the visible spectrum, non-visible spectrum, orboth. In some embodiments, multiple images can be obtained acrossvarious optical frequencies.

In some embodiments, a first bead includes a first optical label andspatial barcodes each having a first spatial barcode sequence. A secondbead includes a second optical label and spatial barcodes each having asecond spatial barcode sequence. The first optical label and secondoptical label can be different (e.g., provided by two differentfluorescent dyes or the same fluorescent dye at two differentintensities). The first and second spatial barcode sequences can bedifferent nucleic acid sequences. In some embodiments, the beads can beimaged to identify the first and second optical labels, and the firstand second optical labels can then be used to associate the first andsecond optical labels with the first and second spatial barcodesequences, respectively. In some embodiments, the nucleic acidcontaining the spatial barcode can further have a capture domain foranalytes (e.g., mRNA). In some embodiments, the nucleic acid (e.g.,nucleic acid containing the spatial barcode) can have a unique molecularidentifier, a cleavage domain, a functional domain, or combinationsthereof.

In some embodiments, the optical label has a characteristicelectromagnetic spectrum. As used herein, the “electromagnetic spectrum”refers to the range of frequencies of electromagnetic radiation. In someembodiments, the optical label has a characteristic absorption spectrum.As used herein, the “absorption spectrum” refers to the range offrequencies of electromagnetic radiation that are absorbed. The“electromagnetic spectrum” or “absorption spectrum” can lead todifferent characteristic spectrum. In some embodiments, the peakradiation or the peak absorption occurs at 380-450 nm (Violet), 450-485nm (Blue), 485-500 nm (Cyan), 500-565 nm (Green), 565-590 nm (Yellow),590-625 nm (Orange), or 625-740 nm (Red). In some embodiments, the peakradiation or the peak absorption occurs around 400 nm, 460 nm, or 520nm.

Optical labels included on the beads can identify the associated spatialbarcode on the bead. Due to the relative limited diversity of opticallabels it can be advantageous to limit the size of the spatial array fordeconvolution. For example, the substrate can be partitioned into two ormore partitions (e.g., bins). In some embodiments, the substrate can bepartitioned into three or more partitions. In some embodiments, thesubstrate can be partitioned into four or more partitions (e.g., bins).In some embodiments, a set of beads are deposited to the partition.Within each set of beads, one or more beads (e.g., equal to or more than10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000 beads) canhave an unique optical label.

In some cases, beads within the same partition can have differentcoordinates on the substrate. These coordinates can be determined e.g.,by various imaging techniques, such as observation through microscopeunder an appropriate condition. In some embodiments, the beads withinthe same partition can share the same spatial barcode. In someembodiments, the beads (e.g., beads having capture probes with barcodes,e.g., spatial barcodes or UMI) are different from each other fordifferent partition bins. In some embodiments, the beads having captureprobes with barcodes (e.g., spatial barcodes or UMI) can have differentbarcodes. For example, in some cases, within each set of beads, whichbeads are associated with a capture probe, the capture probes onindividual beads can have a unique barcode. In some cases, among allbeads (e.g., within two or more sets of beads), individual beads canhave capture probes with a unique barcode.

In some aspects, the present disclosure provides a substrate. Thesubstrate can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300,400, 500, 600, 700, 800, 900, 1000, or more than 1000 partitions (e.g.,bins, or pre-defined area). The partitions can have the same shape ordifferent shapes. In some embodiments, the substrate has only onepartition (e.g., bin or pre-defined area).

In some embodiments, the first partition (e.g., the first pre-definedarea, or the only bin on the substrate) can have a first set of beads.In some embodiments, at least one bead from the first set of beadscomprises an optical label, and a capture probe (e.g., anoligonucleotide capture probe) comprising a barcode and a capturedomain. At least one of the beads can have a unique optical label amongthe first set of beads. In some embodiments, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% ofthe beads in the first set of beads have a unique optical label. In someembodiments, each bead in the first set of beads has a unique opticallabel.

In some embodiments, the substrate can have a second partition (e.g.,the second pre-defined area, or the second bin). The second partitioncan have a second set of beads. In some embodiments, at least one beadfrom the second set of beads comprises an optical label, and a captureprobe (e.g., an oligonucleotide capture probe) comprising a barcode anda capture domain. At least one of the beads can have a unique opticallabel among the second set of beads. In some embodiments, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% of the beads in the second set of beads have a unique opticallabel. In some embodiments, each bead in the second set of beads has aunique optical label.

In some embodiments, the substrate can have a third partition, a fourthpartition, a fifth partition, a sixth partition, a seventh partition, aneighth partition, a ninth partition, or a tenth partition, etc. In someembodiments, the substrate can have multiple partitions. In some cases,each of these partitions has properties that are similar to the first orthe second partitions described herein. For example, at least one beadfrom each set of beads comprises an optical label, and a capture probe(e.g., an oligonucleotide capture probe) comprising a barcode and acapture domain. At least one of these beads can have a unique opticallabel among each set of beads. In some embodiments, at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%of the beads in each set of beads have a unique optical label. In someembodiments, each bead in each set of beads has a unique optical label.

In some embodiments, the beads are deposited on the substrate. In someembodiments, the beads can be deposited directly on or into a biologicalsample. Thus, in some cases, the biological sample can be fixed orattached on the substrate before beads are deposited onto the substrate.

In some embodiments, the beads are only deposited to areas of interest(e.g., specific locations on the substrate, specific cell types, andspecific tissue structures). Thus, the deposited beads do notnecessarily cover the entire biological sample. In some embodiments, oneor more regions of a substrate can be masked or modified (e.g., cappedcapture domains) such that the masked regions do not interact with acorresponding region of the biological sample.

In some embodiments, two or more than two sets of beads (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 sets) are deposited at two or morethan two partitions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10partitions). These partitions do not need to be adjacent to each other.As long as the location of the partitions on the substrate is recorded,the identity of the beads can be determined from the optical labels.

In some embodiments, a set of beads can have equal to or more than 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, or 5000 beads. In some embodiments, a set 25 of beads can haveless than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, or 5000 beads.

Optical labels can be included while generating the beads. For example,optical labels can be included in the polymer structure of a gel bead,or attached at the pre-polymer or monomer stage in bead production. Insome embodiments, the beads include moieties that attach to one or moreoptical labels (e.g., at a surface of a bead and/or within a bead). Insome embodiments, optical labels can be loaded into the beads with oneor more reagents. For example, reagents and optical labels can be loadedinto the beads by diffusion of the reagents (e.g., a solution ofreagents including the optical labels). In some embodiments, opticallabels can be included while preparing spatial barcodes. For example,spatial barcodes can be prepared by synthesizing molecules includingbarcode sequences (e.g., using a split pool or combinatorial approach).Optical labels can be attached to spatial barcodes prior to attachingthe spatial barcodes to a bead. In some embodiments, optical labels canbe included after attaching spatial barcodes to a bead. For example,optical labels can be attached to spatial barcodes coupled to the bead.In some embodiments, spatial barcodes or sequences thereof can bereleasably or cleavably attached to the bead. Optical labels can bereleasably or non-releasably attached to the bead. In some embodiments,a first bead (e.g., a bead including a plurality of spatial barcodes)can be coupled to a second bead including one or more optical labels.For example, the first bead can be covalently coupled to the second beadvia a chemical bond. In some embodiments, the first bead can benon-covalently associated with the second bead.

The first and/or second bead can include a plurality of spatialbarcodes. The plurality of spatial barcodes coupled to a given bead caninclude the same barcode sequences. Where both the first and secondbeads include spatial barcodes, the first and second beads can includespatial barcodes including the same barcode sequences or differentbarcode sequences.

Bead arrays containing captured analytes can be processed in bulk orpartitioned into droplet emulsions for preparing sequencing libraries.In some embodiments, next generation sequencing reads are clustered andcorrelated to the spatial position of the spatial barcode on the beadarray. For example, the information can be computationally superimposedover a high-resolution image of the tissue section to identify thelocation(s), where the analytes were detected.

In some embodiments, de-cross linking can be performed to account forde-crosslinking chemistries that may be incompatible with certainbarcoding/library prep biochemistry (e.g., presence of proteases). Forexample, a two-step process is possible. In the first step, beads can beprovided in droplets such that DNA binds to the beads after theconventional de-crosslinking chemistry is performed. In the second step,the emulsion is broken and beads collected and then re-encapsulatedafter washing for further processing.

In some embodiments, beads can be affixed or attached to a substrateusing photochemical methods. For example, a bead can be functionalizedwith perfluorophenylazide silane (PFPA silane), contacted with asubstrate, and then exposed to irradiation (see, e.g., Liu et al. (2006)Journal of the American Chemical Society 128, 14067-14072). For example,immobilization of antraquinone-functionalized substrates (see, e.g.,Koch et al. (2000) Bioconjugate Chem. 11, 474-483, the entire contentsof which are herein incorporated by reference).

The arrays can also be prepared by bead self-assembly. Each bead can becovered with hundreds of thousands of copies of a specificoligonucleotide. In some embodiments, each bead can be covered withabout 1,000 to about 1,000,000 oligonucleotides. The beads can beirregularly distributed across etched substrates during the arrayproduction process. During this process, the beads can be self-assembledinto arrays (e.g., on a fiber-optic bundle substrate or a silica slidesubstrate). In some embodiments, the beads irregularly arrive at theirfinal location on the array. Thus, the bead location may need to bemapped or the oligonucleotides may need to be synthesized based on apredetermined pattern.

Beads can be affixed or attached to a substrate covalently,non-covalently, with adhesive, or a combination thereof. The attachedbeads can be, for example, layered in a monolayer, a bilayer, atrilayer, or as a cluster. As defined herein, a “monolayer” generallyrefers to an arrayed series of probes, beads, spots, dots, features,micro-locations, or islands that are affixed or attached to a substrate,such that the beads are arranged as one layer of single beads. In someembodiments, the beads are closely packed.

As defined herein, the phrase “substantial monolayer” or “substantiallyform(s) a monolayer” generally refers to (the formation of) an arrayedseries of probes, beads, microspheres, spots, dots, features,micro-locations, or islands that are affixed or attached to a substrate,such that about 50% to about 99% (e.g., about 50% to about 98%) of thebeads are arranged as one layer of single beads. This arrangement can bedetermined using a variety of methods, including microscopic imaging.

In some embodiments, the monolayer of beads is a located in a predefinedarea on the substrate. For example, the predefined area can bepartitioned with physical barriers, a photomask, divots in thesubstrate, or wells in the substrate.

As used herein, the term “reactive element” generally refers to amolecule or molecular moiety that can react with another molecule ormolecular moiety to form a covalent bond. Reactive elements include, forexample, amines, aldehydes, alkynes, azides, thiols, haloacetyls,pyridyl disulfides, hydrazides, carboxylic acids, alkoxyamines,sulfhydryls, maleimides, Michael acceptors, hydroxyls, and activeesters. Some reactive elements, for example, carboxylic acids, can betreated with one or more activating agents (e.g., acylating agents,isourea-forming agents) to increase susceptibility of the reactiveelement to nucleophilic attack. Non-limiting examples of activatingagents include N-hydroxysuccinimide, N-hydroxysulfosuccinimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide,diisopropylcarbodiiimide, 1-hydroxybenzotriazole,(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexfluorophosphate,(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate,4-(N,N-dimethylamino)pyridine, and carbonyldiimidazole.

In some embodiments, the reactive element is bound directly to a bead.For example, hydrogel beads can be treated with an acrylic acid monomerto form acrylic acid—functionalized hydrogel beads. In some cases, thereactive element is bound indirectly to the bead via one or morelinkers. As used herein, a “linker” generally refers to amultifunctional (e.g., bifunctional, trifunctional) reagent used forconjugating two or more chemical moieties. A linker can be a cleavablelinker that can undergo induced dissociation. For example, thedissociation can be induced by a solvent (e.g., hydrolysis andsolvolysis); by irradiation (e.g., photolysis); by an enzyme (e.g.,enzymolysis); or by treatment with a solution of specific pH (e.g., pH4, 5, 6, 7, or 8).

In some embodiments, the reactive element is bound directly to asubstrate. For example, a glass slide can be coated with(3-aminopropyl)triethoxysilane. In some embodiments, the reactiveelement is bound indirectly to a substrate via one or more linkers.

Gel/Hydrogel Beads

In some embodiments, the bead can be a gel bead. A “gel” is a semi-rigidmaterial permeable to liquids and gases. Exemplary gels include, but arenot limited to, those having a colloidal structure, such as agarose;polymer mesh structures, such as gelatin; hydrogels; and cross-linkedpolymer structures, such as polyacrylamide, SFA (see, for example, U.S.Patent Application Publication No. 2011/0059865, which is incorporatedherein by reference in its entirety) and PAZAM (see, for example, U.S.Patent Application Publication No. 2014/0079923, which is incorporatedherein by reference in its entirety).

A gel can be formulated into various shapes and dimensions depending onthe context of intended use. In some embodiments, a gel is prepared andformulated as a gel bead (e.g., a gel bead including capture probesattached or associated with the gel bead). A gel bead can be a hydrogelbead. A hydrogel bead can be formed from molecular precursors, such as apolymeric or monomeric species.

In some cases, a bead comprises a polymer or hydrogel. The polymer orhydrogel may determine one or more characteristics of the hydrogel bead,such as the volume, fluidity, porosity, rigidity, organization, or oneor more other features of the hydrogel bead. In some embodiments, ahydrogel bead can include a polymer matrix (e.g., a matrix formed bypolymerization or cross-linking). A polymer matrix can include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Cross-linking can be via covalent, ionic, and/orinductive interactions, and/or physical entanglement.

A polymer or hydrogel may be formed, for example, upon cross-linking oneor more cross-linkable molecules within the hydrogel bead. For example,a hydrogel may be formed upon cross-linking one or more molecules withinthe hydrogel bead. The hydrogel may be formed upon polymerizing aplurality of monomers within the hydrogel bead. The hydrogel may beformed upon polymerizing a plurality of polymers within the hydrogelbead. Polymeric or hydrogel precursors may be provided to the hydrogelbead and may not form a polymer or hydrogel without application of astimulus (e.g., as described herein). In some cases, the hydrogel beadmay be encapsulated within the polymer or hydrogel. Formation of ahydrogel bead may take place following one or more other changes to thecell that may be brought about by one or more other conditions.

The methods described herein may be applied to a single hydrogel bead ora plurality of hydrogel beads. A method of processing a plurality ofhydrogel beads may comprise providing the plurality of hydrogel beadswithin a vessel and subjecting the plurality of hydrogel beads toconditions sufficient to change one or more characteristics of thehydrogel bead. For example, plurality of hydrogel beads may be subjectedto a first condition or set of conditions comprising a chemical species,and a cross-section of the hydrogel beads of the plurality of hydrogelbeads may change from a first cross-section to a second cross-section,which second cross-section is less than the first cross-section. Thechemical species may comprise, for example, an organic solvent such asethanol, methanol, or acetone. The plurality of hydrogel beads may thenbe subjected to a second condition or set of conditions comprising achemical species, and crosslinks may form within each of the hydrogelbeads. The chemical species may comprise, for example, a cross-linkingagent. The plurality of processed hydrogel beads may be provided in anaqueous fluid. In some instances, the second cross-section of theplurality of hydrogel beads is substantially maintained in the aqueousfluid. The plurality of processed hydrogel beads may be partitionedwithin a plurality of partitions. The partitions may be, for example,aqueous droplets included in a water-in-oil emulsion. The partitions maybe, for example, a plurality of wells. The plurality of fixed hydrogelbeads may be co-partitioned with one or more reagents. In some cases,the plurality of fixed hydrogel beads may be co-partitioned with one ormore beads, where each bead comprises a plurality of nucleic acidbarcode molecules attached thereto. The nucleic acid barcode moleculesattached to a given bead may comprise a common barcode sequence, and thenucleic acid barcode molecules attached to each different bead maycomprise a sequence comprising a different common barcode sequence. Thenucleic acid barcode molecules, or portions thereof, may then be used inreactions with target molecules associated with hydrogel beads of theplurality of hydrogel beads.

(2) Methods for Covalently Bonding Features to a Substrate

Provided herein are methods for the covalent bonding of features (e.g.,optically labeled beads, hydrogel beads, microsphere beads) to asubstrate.

In some embodiments, the features (e.g., beads) are coupled to asubstrate via a covalent bond between a first reactive element and asecond reactive element. In some embodiments, the covalently-bound beadssubstantially form a monolayer of features (e.g., hydrogel beads,microsphere beads) on the substrate.

In some embodiments, the features (e.g., beads) are functionalized witha first reactive element, which is directly bound to the features. Insome embodiments, the features are functionalized with a first reactiveelement, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. In some embodiments, thelinker is an amino methacrylamide. For example, the linker can be3-aminopropyl methacrylamide. In some embodiments, the linker is a PEGlinker. In some embodiments, the linker is a cleavable linker.

In some embodiments, the substrate is functionalized with a secondreactive element, which is directly bound to the substrate. In someembodiments, the substrate is functionalized with a second reactiveelement, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. For example, the linker canbe benzophenone. In some embodiments, the linker is an aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In someembodiments, the linker is a cleavable linker.

In some embodiments, the substrate is a glass slide. In someembodiments, the substrate is a pre-functionalized glass slide.

(3) Methods for Non-Covalently Bonding Features to a Substrate

Provided herein are methods for the non-covalent bonding of features(e.g., optically-labeled beads, hydrogel beads, or microsphere beads) toa substrate.

In some embodiments, features (e.g., beads) are coupled to a substratevia a non-covalent bond between a first affinity group and a secondaffinity group. In some embodiments, the non-covalently-bound features(e.g., beads) substantially form a monolayer of beads (e.g., hydrogelbeads, microsphere beads) on the substrate.

In some embodiments, the features are functionalized with a firstaffinity group, which is directly bound to the features. In someembodiments, the features are functionalized with a first affinitygroup, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. In some embodiments, thelinker is an amino methacrylamide. For example, the linker can be3-aminopropyl methacrylamide. In some embodiments, the linker is a PEGlinker. In some embodiments, the linker is a cleavable linker.

In some embodiments, the substrate is functionalized with a secondaffinity group, which is directly bound to the substrate. In someembodiments, the substrate is functionalized with a second affinitygroup, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. In some embodiments, thelinker is an amino methacrylamide. For example, the linker can be3-aminopropyl methacrylamide. In some embodiments, the linker is a PEGlinker. In some embodiments, the linker is a cleavable linker.

In some embodiments the first affinity group or the second affinitygroup is biotin, and the other of the first affinity group or the secondaffinity group is streptavidin.

In some embodiments, the monolayer of beads is a formed in a predefinedarea on the substrate. In some embodiments, the predefined area ispartitioned with physical barriers. For example, divots or wells in thesubstrate. In some embodiments, the predefined area is partitioned usinga photomask. For example, the substrate is coated with a photo-activatedsolution, dried, and then irradiated under a photomask. In someembodiments, the photo-activated solution is UV-activated.

As used herein, an “adhesive” generally refers to a substance used forsticking objects or materials together. Adhesives include, for example,glues, pastes, liquid tapes, epoxy, bioadhesives, gels, and mucilage. Insome embodiments, an adhesive is liquid tape. In some embodiments, theadhesive is glue.

In some embodiments, beads are adhered to a substrate using an adhesive(e.g., liquid tape, glue, paste). In some embodiments, the adhered beadssubstantially form a monolayer of beads on the substrate (e.g., a glassslide). In some embodiments, the beads are hydrogel beads. In someembodiments, the beads are microsphere beads. In some embodiments, thebeads are coated with the adhesive, and then the beads are contactedwith the substrate. In some embodiments, the substrate is coated withthe adhesive, and then the substrate is contacted with the beads. Insome embodiments, both the substrate is coated with the adhesive and thebeads are coated with the adhesive, and then the beads and substrate arecontacted with one another.

In some embodiments, beads can be deposited onto a biological samplesuch that the deposited beads form a monolayer of beads on thebiological sample (e.g., over or under the biological sample). In someembodiments, beads deposited on the substrate can self-assemble into amonolayer of beads that saturate the intended surface area of thebiological sample under investigation. In this approach, bead arrays canbe designed, formulated, and prepared to evaluate a plurality ofanalytes from a biological sample of any size or dimension. In someembodiments, the concentration or density of beads (e.g., gel beads)applied to the biological sample is such that the area as a whole, orone or more regions of interest in the biological sample, is saturatedwith a monolayer of beads. In some embodiments, the beads are contactedwith the biological sample by pouring, pipetting, spraying, and thelike, onto the biological sample. Any suitable form of bead depositioncan be used.

In some embodiments, the biological sample can be confined to a specificregion or area of the array. For example, a biological sample can beaffixed to a glass slide and a chamber, gasket, or cage positioned overthe biological sample to act as a containment region or frame withinwhich the beads are deposited. As will be apparent, the density orconcentration of beads needed to saturate an area or biological samplecan be readily determined by one of ordinary skill in the art (e.g.,through microscopic visualization of the beads on the biologicalsample). In some embodiments, the bead array contains microfluidicchannels to direct reagents to the spots or beads of the array.

(4) Feature Geometric Attributes

Features on an array can have a variety of sizes. In some embodiments, afeature of an array can have an average diameter or maximum dimensionbetween 500 nm μm to 100 μm. For example, between 500 nm to 2 μm, 1 μmto 3 μm, 1 μm to 5 μm, 1 μm to 10 μm, 1 μm to 20 μm, 1 μm to 30 μm, 1 μmto 40 μm, 1 μm to 50 μm, 1 μm to 60 μm, 1 μm to 70 μm, 1 μm to 80 μm, 1μm to 90 μm, 90 μm to 100 μm, 80 μm to 100 μm, 70 μm to 100 μm, 60 μm to100 μm, 50 μm to 100 μm, 40 μm to 100 μm, 30 μm to 100 μm, 20 μm to 100μm, 10 μm to 100 μm, about 40 μm to about 70 μm, or about 50 μm to about60 μm. In some embodiments, the feature has an average diameter ormaximum dimension between 30 μm to 100 μm, 40 μm to 90 μm, 50 μm to 80μm, 60 μm to 70 μm, or any range within the disclosed sub-ranges. Insome embodiments, the feature has an average diameter or maximumdimension no larger than 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4μm, 3 μm, 2 μm, or 1 μm. In some embodiments, the feature has an averagediameter or maximum dimension of approximately 65 μm. In someembodiments, the feature has an average diameter or maximum distance ofapproximately 55 μm.

In some embodiments, the size and/or shape of a plurality of features ofan array are approximately uniform. In some embodiments, the size and/orshape of a plurality of features of an array is not uniform. Forexample, in some embodiments, features in an array can have an averagecross-sectional dimension, and a distribution of cross-sectionaldimensions among the features can have a full-width and half-maximumvalue of 0% or more (e.g., 5% or more, 10% or more, 20% or more, 30% ormore, 40% or more, 50% or more, 70% or more, or 100% or more) of theaverage cross-sectional dimension for the distribution.

In certain embodiments, features in an array can have an averagecross-sectional dimension of between about 1 μm and about 10 μm. Thisrange in average feature cross-sectional dimension corresponds to theapproximate diameter of a single mammalian cell. Thus, an array of suchfeatures can be used to detect analytes at, or below, mammaliansingle-cell resolution.

In some embodiments, a plurality of features has a mean diameter or meanmaximum dimension of about 0.1 μm to about 100 μm (e.g., about 0.1 μm toabout 5 μm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm toabout 80 μm, about 1 μm to about 90 μm, about 90 μm to about 100 μm,about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm toabout 100 μm, about 50 μm to about 100 μm, about 40 μm to about 100 μm,about 30 μm to about 100 μm, about 20 μm to about 100 μm, or about 10 μmto about 100 μm). In some embodiments, the plurality of features has amean diameter or mean maximum dimension between 30 μm to 100 μm, 40 μmto 90 μm, 50 μm to 80 μm, 60 μm to 70 μm, or any range within thedisclosed sub-ranges. In some embodiments, the plurality of features hasa mean diameter or a mean maximum dimension no larger than 95 μm, 90 μm,85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35μm, 30 μm, 25 μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm,8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm. In some embodiments,the plurality of features has a mean average diameter or a mean maximumdimension of approximately 65 μm, approximately 60 μm, approximately 55μm, approximately 50 μm, approximately 45 μm, approximately 40 μm,approximately 35 μm, approximately 30 μm, approximately 25 μm,approximately 20 μm, approximately 15 μm, approximately 10 μm,approximately 5 μm, approximately 4 μm, approximately 3 μm,approximately 2 μm, or approximately 1 μm.

(iii) Array Geometric Attributes

In some embodiments, an array includes a plurality of features. Forexample, an array includes between 4,000 and 50,000 features, or anyrange within 4,000 to 40,000 features. For example, an array includesbetween 4,000 to 35,000 features, 4,000 to 30,000 features, 4,000 to25,000 features, 4,000 to 20,000 features, 4,000 to 15,000 features,4,000 to 10,000 features, 4,000 to 6,000 features, or 4,400 to 6,000features. In some embodiments, the array includes between 4,100 and5,900 features, between 4,200 and 5,800 features, between 4,300 and5,700 features, between 4,400 and 5,600 features, between 4,500 and5,500 features, between 4,600 and 5,400 features, between 4,700 and5,300 features, between 4,800 and 5,200 features, between 4,900 and5,100 features, or any range within the disclosed sub-ranges. Forexample, the array can include about 4,000 features, about 4,200features, about 4,400 features, about 4,800 features, about 5,000features, about 5,200 features, about 5,400 features, about 5,600features, or about 6,000 features, about 10,000 features, about 20,000features, about 30,000 features, about 40,000 features, or about 50,000features. In some embodiments, the array comprises at least 4,000features. In some embodiments, the array includes approximately 5,000features.

In some embodiments, features within an array have an irregulararrangement or relationship to one another, such that no discernablepattern or regularity is evident in the geometrical spacingrelationships among the features. For example, features within an arraymay be positioned randomly with respect to one another. Alternatively,features within an array may be positioned irregularly, but the spacingsmay be selected deterministically to ensure that the resultingarrangement of features is irregular.

In some embodiments, features within an array are positioned regularlywith respect to one another to form a pattern. A wide variety ofdifferent patterns of features can be implemented in arrays. Examples ofsuch patterns include, but are not limited to, square arrays offeatures, rectangular arrays of features, hexagonal arrays of features(including hexagonal close-packed arrays), radial arrays of features,spiral arrays of features, triangular arrays of features, and moregenerally, any array in which adjacent features in the array are reachedfrom one another by regular increments in linear and/or angularcoordinate dimensions.

In some embodiments, features within an array are positioned with adegree of regularity with respect to one another such that the array offeatures is neither perfectly regular nor perfectly irregular (i.e., thearray is “partially regular”). For example, in some embodiments,adjacent features in an array can be separated by a displacement in oneor more linear and/or angular coordinate dimensions that is 10% or more(e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more,70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120%or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% ormore, 180% or more, 190% or more, 200% or more) of an averagedisplacement or a nominal displacement between adjacent features in thearray. In certain embodiments, the distribution of displacements (linearand/or angular) between adjacent features in an array has a full-widthat half-maximum of between 0% and 200% (e.g., between 0% and 100%,between 0% and 75%, between 0% and 50%, between 0% and 25%, between 0%and 15%, between 0% and 10%) of an average displacement or nominaldisplacement between adjacent features in the array.

In some embodiments, arrays of features can have a variable geometry.For example, a first subset of features in an array can be arrangedaccording to a first geometrical pattern, and a second subset offeatures in the array can be arranged according to a second geometricalpattern that is different from the first pattern. Any of the patternsdescribed above can correspond to the first and/or second geometricalpatterns, for example.

In general, arrays of different feature densities can be prepared byadjusting the spacing between adjacent features in the array. In someembodiments, the geometric center-to-center (e.g., pitch) spacingbetween adjacent features in an array is between 100 nm to 10 μm, 500 nmto 2 μm, 1 μm to 5 μm, and 20 μm to 200 μm. For example, thecenter-to-center spacing can be between 100 nm to 10 μm, 500 nm to 2 μm,1 μm, to 5 μm, 20 μm to 40 μm, 20 μm to 60 μm, 20 μm to 80 μm, 80 μm to100 μm, 100 μm to 120 μm, 120 μm to 140 μm, 140 μm to 160 μm, 160 μm to180 μm, 180 μm to 200 μm, 60 μm to 100 μm, or 40 μm to 100 μm, 50 μm to150 μm, 80 μm to 120 μm, or 90 μm to 110 μm. In some embodiments, thepitch between adjacent array features is between 30 μm and 100 μm, 40 μmand 90 μm, 50 μm and 80 μm, 60 μm and 70 μm, 80 μm and 120 μm, or anyrange within the disclosed sub-ranges. In some embodiments, the pitchbetween adjacent array features of an array is approximately 65 μm,approximately 60 μm, approximately 55 μm, approximately 50 μm,approximately 45 μm, approximately 40 μm, approximately 35 μm,approximately 30 μm, approximately 25 μm, approximately 20 μm,approximately 15 μm, approximately 10 μm, approximately 5 μm,approximately 4 μm, approximately 3 μm, approximately 2 μm, orapproximately 1 μm. In some embodiments, the pitch between adjacentarray features of an array is less than 100 μm.

An array of features can have any appropriate resolution. In someembodiments, an array of features can have a spatially constant (e.g.,within a margin of error) resolution. In general, an array with aspatially consistent resolution is an array in which the pitch betweenadjacent features in the array is constant (e.g., within a margin oferror). Such arrays can be useful in a variety of applications. In someembodiments, an array of features can have a spatially varyingresolution. In general, an array with a spatially varying resolution isan array in which the center-to-center spacing (e.g., pitch) (alonglinear, angular, or both linear and angular coordinate dimensions)between adjacent features in the array varies. Such arrays can be usefulin a variety of applications. For example, in some embodiments,depending upon the spatial resolution at which the sample is to beinvestigated, the sample can be selectively associated with the portionof the array that corresponds approximately to the desired spatialresolution of the measurement.

In some embodiments, it may be useful to describe the resolution of anarray of features by functional aspects, for example, the number ofreads that can be carried out per feature (which can be a proxy forsequencing saturation), the number of transcripts that can be detectedper feature, or the number of genes that can be detected per feature.For example, in some embodiments, the number of reads that can beperformed per feature is between 50,000 and 1,000,000. For example, thenumber of reads that can be performed per feature can be between 50,000and 100,000, 50,000 and 150,000, 50,000 and 200,000, 50,000 and 250,000,50,000 and 300,000, 50,000 and 350,000, 50,000 and 400,000, 50,000 and500,000, 50,000 and 550,000, 50,000 and 600,000, 50,000 and 650,000,50,000 and 700,000, 50,000 and 750,000, 50,000 and 800,000, 50,000 and850,000, 50,000 and 900,000, 50,000 and 950,000, 50,000 and 1,000,000,100,000 to 500,000, 150,000 to 500,000, 200,000 to 500,000, 250,000 to500,000, 300,000 and 500,000, 350,000 and 500,000, 400,000 and 500,000,450,000 and 500,000, 150,000 to 250,000, or 300,000 to 400,000.

In some embodiments, the number of transcripts that can be detected perfeature is between 20,000 and 200,000. For example, in some embodiments,the number of transcripts that can be detected per feature can bebetween 20,000 and 30,000, 20,000 and 40,000, 20,000 and 50,000, 30,000and 60,000, 40,000 and 60,000, 50,000 and 60,000, 20,000 and 100,000,30,000 and 100,000, 40,000 and 200,000, 50,000 and 200,000, or 30,000and 200,000.

In some embodiments, the number of genes that can be detected perfeature is between 1,000 and 5,000. For example, the number of genesthat can be detected per feature can be between 1,000 and 1,500, 1,000and 2,000, 1,000 and 2,500, 1,000 and 3,000, 1,000 and 3,500, 1,000 and4,000, 1,000 and 4,500, 1,500 and 5,000, 2,000 and 5,000, 2,500 and5,000, 3,000 and 5,000, 3,500 and 5,000, 4,000 and 5,000, 4,500 and5,000, 1,500 and 2,500, 2,500 and 3,500, or 3,500 and 4,000.

In some embodiments, it may be useful to describe the resolution of anarray of features by functional aspects, for example, the number of UMIcounts per feature. For example, in some embodiments, the number of UMIcounts that can be performed per feature is between 1,000 and 50,000. Insome embodiments, the number of UMI counts can be averaged to obtain amean UMI per feature. In some embodiments, the number of UMI counts canbe averaged to obtain a median UMI count per feature. For example, themedian UMI count per feature can be between 1,000 and 50,000, 1,000 and40,000, 1,000 and 30,000, 1,000 and 20,000, 1,000 and 10,000, 1,000 and5,000.

These components can be used to determine the sequencing saturation ofthe array. The sequencing saturation can be a measure of the librarycomplexity and sequencing depth. For example, different cell types willhave different amounts of RNA, thus different number of transcripts,influencing library complexity. Additionally, sequencing depth isrelated to the number of sequencing reads. In some embodiments, theinverse of sequencing saturation is the number of additional reads itwould take to detect a new transcript. One way of measuring thesequencing saturation of an array is to determine the number of reads todetect a new UMI. For example, if a new UMI is detected every 2 reads ofthe feature, the sequencing saturation would be 50%. As another example,if a new UMI is detected every 10 reads of a feature, the sequencingsaturation would be 90%.

Arrays of spatially varying resolution can be implemented in a varietyof ways. In some embodiments, for example, the pitch between adjacentfeatures in the array varies continuously along one or more linearand/or angular coordinate directions. Thus, for a rectangular array, thespacing between successive rows of features, between successive columnsof features, or between both successive rows and successive columns offeatures, can vary continuously.

In certain embodiments, arrays of spatially varying resolution caninclude discrete domains with populations of features. Within eachdomain, adjacent features can have a regular pitch. Thus, for example,an array can include a first domain within which adjacent features arespaced from one another along linear and/or angular coordinatedimensions by a first set of uniform coordinate displacements, and asecond domain within which adjacent features are spaced from one anotheralong linear and/or angular coordinate dimensions by a second set ofuniform coordinate displacements. The first and second sets ofdisplacements differ in at least one coordinate displacement, such thatadjacent features in the two domains are spaced differently, and theresolution of the array in the first domain is therefore different fromthe resolution of the array in the second domain.

In some embodiments, the pitch of array features can be sufficientlysmall such that array features are effectively positioned continuouslyor nearly continuously along one or more array dimensions, with littleor no displacement between array features along those dimensions. Forexample, in a feature array where the features correspond to regions ofa substrate (i.e., oligonucleotides are directly bound to thesubstrate), the displacement between adjacent oligonucleotides can bevery small—effectively, the molecular width of a single oligonucleotide.In such embodiments, each oligonucleotide can include a distinct spatialbarcode such that the spatial location of each oligonucleotide in thearray can be determined during sample analysis. Arrays of this type canhave very high spatial resolution, but may only include a singleoligonucleotide corresponding to each distinct spatial location in asample.

In general, the size of the array (which corresponds to the maximumdimension of the smallest boundary that encloses all features in thearray along one coordinate direction) can be selected as desired, basedon criteria such as the size of the sample, the feature diameter, andthe density of capture probes within each feature. For example, in someembodiments, the array can be a rectangular or square array for whichthe maximum array dimension along each coordinate direction is 10 mm orless (e.g., 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mmor less, 4 mm or less, 3 mm or less). Thus, for example, a square arrayof features can have dimensions of 8 mm by 8 mm, 7 mm by 7 mm, 5 mm by 5mm, or be smaller than 5 mm by 5 mm.

(iv) Bead Arrays

As used herein, the term “bead array” refers to an array that includes aplurality of beads as the features in the array. In some embodiments,two or more beads are dispersed onto a substrate to create an array,where each bead is a feature on the array. In some embodiments, thebeads are attached to a substrate. For example, the beads can optionallyattach to a substrate such as a microscope slide and in proximity to abiological sample (e.g., a tissue section that includes cells). Thebeads can also be suspended in a solution and deposited on a surface(e.g., a membrane, a tissue section, or a substrate (e.g., a microscopeslide)). Beads can optionally be dispersed into wells on a substrate,e.g., such that only a single bead is accommodated per well.

Examples of arrays of beads on or within a substrate include beadslocated in wells such as the BeadChip array for microarray genotyping(available from Illumina Inc., San Diego, Calif.), and array used insequencing platforms from Ion Torrent (a subsidiary of LifeTechnologies, Carlsbad, Calif.). Examples of bead arrays are describedin, e.g., U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570;6,210,891; 6,258,568; and 6,274,320; U.S. Pat. Application PublicationNos. 2009/0026082; 2009/0127589; 2010/0137143; 2019/0177777; and2010/0282617; and PCT Patent Application Publication Nos. WO 00/063437and WO 2016/162309, the entire contents of each of which is incorporatedherein by reference.

In some embodiments, the bead array includes a plurality of beads. Forexample, the bead array can include at least 10,000 beads (e.g., atleast 100,000 beads, at least 1,000,000 beads, at least 5,000,000 beads,at least 10,000,000 beads). In some embodiments, the plurality of beadsincludes a single type of bead (e.g., substantially uniform in volume,shape, and other physical properties, such as translucence). In someembodiments, the plurality of beads includes two or more types ofdifferent beads.

Bead arrays can be generated by attaching beads (e.g., barcoded beads)to a substrate in a regular pattern, or an irregular arrangement. Insome embodiments, the barcode sequences are known before attaching themto the substrate. In some embodiments, the barcode sequences are notknown before attaching them to the substrate. Beads can be attached toselective regions on a substrate by, e.g., selectively activatingregions on the substrate to allow for attachment of the beads.Activating selective regions on the substrate can include activating ordegrading a coating (e.g., a removable coating as described herein) atthe selective regions where the coating has been applied on thesubstrate, rendering the selective regions more permissive to beadattachment as compared to regions outside of the selected regions. Theregions that are rendered more permissive for bead attachment can beconfigured to fit only one bead or multiple beads (e.g., limited by wellsize or surface patterning, such as fabrication techniques). Beads boundto the selected regions can form a two-dimensional array on thesubstrate. The substrate can be uniformly or non-uniformly coated withthe coating. The beads can be any suitable beads described herein,including beads that are attached to one or more spatial barcodes. Beadscan be attached to the selected regions according to any of the methodssuitable for attaching beads to substrates described herein, such asthrough covalent bonds, non-covalent bonds, or chemical linkers.

Any variety of suitable patterning techniques can be used to attachbeads to a substrate surface. In some embodiments, in a non-limitingway, physical techniques such as inkjet printing, optical andoptoelectronic cell trapping, laser-based patterning, acousticpatterning, dielectrophoresis, or magnetic techniques can be used topattern the substrate. Alternatively, chemical and/or physiochemicaltechniques can be used such as, in a non-limiting way, surface chemistrymethods, microcontact printing, microwells and filtration, DUVpatterning, or patterning in microfluidic devices combined withmicrocontact printing (See, e.g., Martinez-Rivas, A., Methods ofmicropatterning and manipulation of cells for biomedical applications,Micromachines (Basel) 8, (2017), which in is incorporated herein byreference).

The coating can be photoreactive, and selectively activating ordegrading the coating involves exposing selected regions of the coatingto light or radiation. Selectivity can be achieved through theapplication of photomasks. Regions of the coating that are exposed tolight can be rendered more permissive for bead attachment (e.g., moreadhesive), as compared to regions not exposed to light (e.g., regionsprotected from the light by a photomask). When applied to the substrate,the beads thus preferentially attach to the more permissive regions onthe substrate, and un-attached beads can optionally be removed from thesubstrate. The light source and/or the photomask can be adjusted toallow further sites on the substrate to become more permissive for beadattachment, allowing additional beads to be attached at these sites.Alternatively, a different light source, or a different photomask can beapplied. The process of photopatterning thus allows beads to be attachedat pre-determined locations on the substrate, thereby generating a beadarray.

Beads can be attached iteratively, e.g., a subset of the beads can beattached at one time, and the process can be repeated to attach one ormore additional subsets of beads. In some embodiments, the size of theactivated spot (e.g., spot on the substrate) is smaller than the size ofa bead. For example, a bead can be attached to the activated substrate(e.g., spot) such that only a single bead attaches to the activatedsubstrate. In some embodiments, the substrate can be washed to removeunbound beads. In some embodiments, the substrate can be activated in asecond location and a second bead can be attached to the activatedsubstrate surface. This process can be done iteratively to attach beadsto the entire substrate, or a portion thereof. Alternatively, beads canbe attached to the substrate all in one step. Furthermore, methods ofattaching beads to a substrate are known in the art. Any suitable methodcan be used, including, in a non-limiting way, specific chemical bonds,non-specific chemical bonds, linkers, physically trapping the beads(e.g., polymer, hydrogel), or any of the methods described herein.

An exemplary workflow for generating a bead array can includeselectively rendering a first set of one or more selected regions on acoated substrate to be more permissive for bead attachment as comparedto regions outside of the selected regions, applying a plurality ofbeads to the array and allowing the beads to attach to the first set ofselected regions, optionally removing un-attached beads, rendering asecond set of one or more selected regions more permissive to beadattachment as compared to regions outside the second set of selectedregions, applying a plurality of beads and allowing the beads to attachto the second set of selected regions, and optionally removing theun-attached beads. This iterative process can be carried out for anynumber of times to generate a patterned bead array.

Another exemplary process includes activating a first region on a coatedsubstrate and exposing the activated first region to a plurality ofbarcoded beads, so that a first set of one or more beads are bound tothe first region; and activating a second region on the coated substrateand exposing the activated second region to a plurality of barcodedbeads, so that a second set of one or more beads are bound to the secondregion, wherein the first set of one or more beads comprise an identicalfirst oligonucleotide sequence unique to the first region of the surfaceof the substrate, and the second set of one or more beads comprise anidentical second oligonucleotide sequence unique to the second region ofthe surface of the substrate, and wherein the first and secondoligonucleotide sequences are different. Additional regions on thecoated substrate may be activated and exposed to additional barcodedbeads. Each set of barcoded beads can include an oligonucleotidesequence that is different from all other sets of barcoded beads andthat is unique to the location of the activated region. In someinstances, a set of barcoded beads includes a two-part barcode, wherethe first part is correlated with the location to which the set of beadsare bound, and the second part is correlated with the location of a beadrelative to the beads in the set. For example, the location of a beadwithin a set of beads bound to the substrate can be identified based onthe sequence information of the first and second part barcodes. Thefirst part of the barcode can be identical across beads within the sameset, and can be attached to the beads before or after the beads arebound to the substrate. The second part of the barcode can be differentbetween two beads within the set, and can be attached to the beads(e.g., ligated onto the first part barcode) before or after the beadsare bound to the substrate. In some instances, the second part of thebarcode is attached to the beads after they are bound to the substrate.

Additionally, the first set of one or more beads and the second set ofone or more beads can be different. In other words, the first set of oneor more beads and the second set of one or more beads can have differentsurface chemistries, different compositions (e.g., solid bead, gel bead,silica bead)(e.g., nanoparticles vs microparticles), and/or physicalvolumes. In some embodiments, a third set of one or more beads, a fourthset of one or more beads, a fifth set of one or more beads or more canhave different surface chemistries, different compositions (e.g., solidbead, gel bead, silica bead)(e.g., nanoparticles vs microparticles),and/or physical volumes can be attached to the substrate surface. Themethods may include removing the beads that do not bind to the first,second, and/or any of the additional regions. In some embodiments,removing the beads comprise washing the beads off the surface of thesubstrate. The removing may be carried out after each round of or afterseveral rounds of activating a region (e.g., first, second or additionalregions on the surface of the substrate), and binding of beads to theactivated region. In some instances, each bead is bound to the substrateat a single location. The beads bound to the first, second, andadditional regions can form a two-dimensional array of beads on thesubstrate.

A photoreactive coating can comprise a plurality of photoreactivemolecules, which can undergo a chemical reaction (e.g., hydrolysis,oxidation, photolysis) when exposed to light of certain wavelengths orrange of wavelengths. A photo-reactive molecule can become reactive whenexposed to light and can react with other molecules and form chemicalbonds with other molecules.

The coating can comprise a polymer, and activating selected regions onthe substrate include modifying the polymer at the respective regions.Modifying the polymer includes, for example, photochemically modifyingthe polymer by exposing the polymer to radiation or light. Alternativelyor additionally, modifying the polymer can include chemically modifyingthe polymer by contacting the polymer with one or more chemicalreagents. In some instances, the coating is a hydrogel. In someinstances, the coating comprises a photoreactive polymer. Exemplaryphoto-reactive polymers include poly(ethylene glycol) (PEG)-basedpolymers, poly(L-lysine) (PLL)-based polymer, copolymer comprisingfunctionalized or unfunctionalized units of PEG and PLL (e.g.,poly-L-lysine-grafted-polyethylene glycol (PLL-g-PEG)), andmethacrylated gelatin (GelMA) polymers.

Beads can also be attached to selective regions on a substrate byselectively crosslinking beads to a coating that has been applied on thesubstrate. For example, a plurality of beads can be applied to asubstrate having a photocrosslinkable coating, and upon crosslinking ofa subset of the beads to the coating, the non-cross-linked beads can beremoved, leaving only the cross-linked beads on the substrate. Theprocess can be repeated multiple times. The coating can include aphoto-crosslinkable polymer. Exemplary photo-crosslinkable polymers aredescribed, e.g., in Shirai, Polymer Journal 46:859-865 (2014), Ravve,Photocrosslinkable Polymers, Light-Associated Reactions of SyntheticPolymers. Springer, New York, N.Y. (2006), and Ferreira et al.Photocrosslinkable Polymers for Biomedical Applications, BiomedicalEngineering—Frontiers and Challenges, Prof. Reza Fazel (Ed.), ISBN:978-953-307-309-5 (2011), each of which are herein incorporated byreference in its entirety.

Suitable light sources for activating, degrading or crosslinking thecoating as described herein include, but are not limited to, Ultraviolet(UV) light (e.g., 250-350 nm or 350-460 nm UV light) and visible light(e.g., broad spectrum visible light). A Digital Micromirror Device (DMD)can also be used to provide the light source.

The distance between a first pair of adjacent selected regions accordingto the methods described herein can be the same or different from asecond pair of adjacent selected regions.

Barcoded beads, or beads comprising a plurality of barcoded probes, canbe generated by first preparing a plurality of barcoded probes on asubstrate, depositing a plurality of beads on the substrate, andgenerating probes attached to the beads using the probes on thesubstrate as a template.

Large scale commercial manufacturing methods allow for millions ofoligonucleotides to be attached to an array. Commercially availablearrays include those from Affymetrix (ThermoFisher Scientific).

In some embodiments, arrays can be prepared according to the methods setforth in WO 2012/140224, WO 2014/060483, WO 2016/162309, WO 2017/019456,WO 2018/091676, and WO 2012/140224, and U.S. Patent Application No.2018/0245142. The entire contents of each of the foregoing documents areherein incorporated by reference.

In some embodiments, a bead array is formed when beads are embedded in ahydrogel layer where the hydrogel polymerizes and secures the relativebead positions. The bead-arrays can be pre-equilibrated and combinedwith reaction buffers and enzymes (e.g., reverse-transcription mix). Insome embodiments, the bead arrays can be stored (e.g., frozen) long-term(e.g., days) prior to use.

(v) Flexible Arrays

A “flexible array” includes a plurality of spatially-barcoded featuresattached to, or embedded in, a flexible substrate (e.g., a membrane, ahydrogel, or tape) placed onto, or proximal to, a biological sample. Insome embodiments, a flexible array includes a plurality ofspatially-barcoded features embedded within a hydrogel.

(vi) Shrinking Hydrogel Features/Arrays

As used herein “shrinking” or “reducing the size” of a hydrogel refersto any process causing the hydrogel to physically contract and/or thesize of the hydrogel to decrease in volume. For example, the scaffold ofthe gel may shrink or “implode” upon solvent removal (see, e.g., Longand Williams. Science. 2018; 362(6420):1244-1245, and Oran et al.Science 2018; 362(6420): 1281-1285; each of which is incorporated hereinby reference in its entirety). As another example, the process to shrinkor reduce the volume of a hydrogel may be one that removes water (i.e.,a dehydrating process) from the hydrogel. There are many methods knownto one of skill in the art for shrinking or reducing the volume of ahydrogel. Non-limiting examples of a method to shrink or reduce thevolume of a hydrogel include exposing the hydrogel to one or more of: adehydrating solvent, a salt, heat, a vacuum, lyophilization,desiccation, filtration, air-drying, or combinations thereof.

In some embodiments, a hydrogel bead can be decreased in volume (e.g.,shrunken hydrogel bead) before being attached to or embedded in ahydrogel. In some embodiments, a hydrogel bead can be decreased involume (e.g., shrunken hydrogel bead) after being attached to orembedded in a hydrogel. In some embodiments, one or more hydrogel beadscan be attached to or embedded in a hydrogel. In some embodiments, oneor more hydrogel beads can be decreased in volume (e.g., one or moreshrunken hydrogel beads) before being attached to or embedded in ahydrogel. In some embodiments, one or more hydrogel beads can bedecreased in volume (e.g., one or more shrunken hydrogel beads) afterbeing attached to or embedded in a hydrogel. In some embodiments, one ormore hydrogel beads attached to or embedded in a hydrogel can bedecreased in volume. For example, the one or more hydrogel beads and thehydrogel that the hydrogel beads are attached to or embedded in aredecreased in volume at the same time (e.g., shrunken hydrogelbead-containing hydrogel). In some embodiments, one or more hydrogelbeads attached to or embedded in a hydrogel can be isometricallydecreased in volume.

In some embodiments, one or more hydrogel beads attached to or embeddedin a hydrogel can be decreased in volume from about 3 fold to about 4fold. For example, one or more hydrogel beads attached to or embedded ina hydrogel can be decreased in volume by removing or exchangingsolvents, salts, or water (e.g., dehydration). In another example, oneor more hydrogel beads attached to or embedded in a hydrogel can bedecreased in volume by controlling temperature or pH. See e.g., Ahmed,E. M. J. of Advanced Research. 2015 Mar.; 6(2):105-121, which isincorporated herein by reference in its entirety. In some embodiments,one or more hydrogel beads attached to or embedded in a hydrogel can bedecreased in volume by removing water.

In some embodiments, decreasing the volume of one or more hydrogel beadsattached to or embedded in a hydrogel can increase the spatialresolution of the subsequent analysis of the sample. The increasedresolution in spatial profiling can be determined by comparison of thespatial analysis of the sample using one or more shrunken hydrogel beadsattached to or embedded in a hydrogel with one or more non-shrunkenhydrogel beads attached to or embedded in a hydrogel.

In some embodiments, a hydrogel bead is not decreased in volume. In someembodiments, a hydrogel bead can be decreased in volume (e.g., shrunkenhydrogel bead). In some embodiments, a shrunken hydrogel gel bead isstabilized. For example, the hydrogel bead can be decreased in volume byremoving solvents, salts, or water (e.g., dehydrated, desiccated, dried,exsiccated) from the hydrogel bead to form a shrunken hydrogel bead. Inanother example, the hydrogel bead can be decreased in volume bycontrolling temperature or pH. See e.g., Ahmed, E. M. J. of AdvancedResearch. 2015 March; 6(2):105-121, which is incorporated herein byreference in its entirety. Non-limiting examples of solvents that may beused to form a shrunken hydrogel bead or shrunken hydrogel bead arrayinclude a ketone, such as methyl ethyl ketone (MEK), isopropanol (IPA),acetone, 1-butanol, methanol (MeOH), dimethyl sulfoxide (DMSO),glycerol, propylene glycol, ethylene glycol, ethanol, (k) 1,4-dioxane,propylene carbonate, furfuryl alcohol, N,N-dimethylformamide (DMF),acetonitrile, aldehyde, such as formaldehyde or glutaraldehyde, or anycombinations thereof.

In some embodiments, the hydrogel bead or hydrogel bead array isshrunken or stabilized via a cross-linking agent. For example, thecross-linking agent may comprise disuccinimidyl suberate (DSS),dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA),dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate(DST), and ethylene glycol bis(succinimidyl succinate) (EGS).

In some embodiments, the hydrogel bead or hydrogel bead array isprocessed with salts to form a shrunken hydrogel bead or shrunkenhydrogel bead array.

In some embodiments, the removal of water comprises an acid.

In some embodiments, the removal of water comprises a dehydratingprocess such as heat, a vacuum, lyophilization, desiccation, filtration,and air-drying.

In some embodiments, the hydrogel bead or hydrogel bead array undergoesan alteration in temperature (e.g., an alteration from about 37° C., 38°C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47°C., 48° C., 49° C. to about 50° C., 51° C., 52° C., 53° C., 54° C., 55°C., 56° C., 57° C., 58° C., 59° C. 60° C., 61° C., 62° C., 63° C., 64°C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., or higher, or anytemperature alteration encompassed within these ranges) to form ashrunken hydrogel bead or shrunken hydrogel bead array.

In some embodiments, a hydrogel bead can be decreased in size in lineardimension by about 2 fold, about 3 fold, about 4 fold, about 5 fold,about 6 fold, about 7 fold, about 8 fold, about 9 fold, or any intervalstherein. In some embodiments, a hydrogel bead can be decreased in volumeby about 1 fold, about 5 fold, about 10 fold, about 15 fold, about 20fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about45 fold, about 50 fold, about 55 fold, about 60 fold, about 65 foldabout 70 fold, about 75 fold, about 80 fold, or any intervals therein.In some embodiments, a hydrogel bead can be decreased in size such thatthe hydrogel bead has an average diameter of about 1 μm to about 15 μm.

In some embodiments, a plurality of hydrogel beads can be decreased insize in linear dimension by about 2 fold, about 3 fold, about 4 fold,about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, orany intervals therein. In some embodiments, a plurality of hydrogelbeads can be decreased in size such that the average diameter of ahydrogel bead is about 1 μm to about 15 μm. In some embodiments, aplurality of hydrogel beads can be decreased in volume by about 1 fold,about 5 fold, about 10 fold, about 15 fold, about 20 fold, about 25fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, about50 fold, about 55 fold, about 60 fold, about 65 fold about 70 fold,about 75 fold, about 80 fold, or any intervals therein.

In some embodiments, a plurality of hydrogel beads can be decreased involume such that the hydrogel bead has an average diameter of about 1 μmto about 15 μm.

In some embodiments, one or more hydrogel beads can be decreased involume at the same time. In some embodiments, one or more hydrogel beadscan be decreased in volume at different times. In some embodiments, oneor more hydrogel beads can be assembled into an array before decreasingthe volume of the one or more hydrogel beads. In some embodiments, oneor more hydrogel beads can be assembled into an array after decreasingthe volume of the one or more hydrogel beads. In some embodiments, theone or more shrunken hydrogel beads can be reversibly attached to asubstrate. In some embodiments, the one or more shrunken hydrogel beadscan be irreversibly attached to a substrate. In some embodiments, theone or more shrunken hydrogel beads can be re-expanded. In someembodiments, the one or more shrunken hydrogel beads can beisometrically re-expanded. In some embodiments, the one or more shrunkenhydrogel beads can be re-expanded primarily in the z-dimension. In someembodiments, the one or more shrunken hydrogel beads attached to asubstrate (e.g., reversibly or irreversibly) can be re-expandedprimarily in the z-dimension. In some embodiments, the one or moreshrunken hydrogel beads attached to a substrate (e.g., reversibly orirreversibly) can be isometrically re-expanded primarily in thez-dimension.

In some embodiments, decreasing the volume of the hydrogel bead (e.g.,shrunken hydrogel bead) can increase the spatial resolution of thesubsequent analysis of the sample. The increased resolution in spatialprofiling can be determined by comparison of spatial analysis of thesample using a shrunken hydrogel bead with a non-shrunken hydrogel bead.For example, in some embodiments, the subsequent analysis of the samplecan include any array-based spatial analysis method disclosed herein.

In some embodiments, one or more physical parameters or dimensionsand/or one or more other characteristics of the hydrogel bead may bechanged. For example, a cross-section of the hydrogel bead may bechanged from a first cross-section to a second cross-section. The firstcross-section may be smaller or larger than the second cross-section.Alternatively, or in addition, one or more other characteristics of thehydrogel bead may be changed. For example, the fluidity, density,rigidity, porosity, refractive index, polarity, and/or othercharacteristic of the hydrogel bead or one or more components thereofmay be changed. In a non-limiting example, the hydrogel bead includes ahydrogel. In another example, the hydrogel bead hydrogel may formcrosslinks within the bead. The same or different conditions may be usedto change or affect different characteristics of the hydrogel bead atthe same or different times. In some cases, a first condition or set ofconditions may be used to change a first characteristic or set ofcharacteristics of the hydrogel bead (e.g., a cross-section) and asecond condition or set of conditions may be used to change a secondcharacteristic or set of characteristics of the hydrogel bead. The firstcondition or set of conditions may be applied at the same or a differenttime as the second condition or set of conditions. For example, a firstcharacteristic or set of characteristics may be changed under a firstcondition or set of conditions, after which a second characteristic orset of characteristics may be changed under a second condition or set ofconditions.

A characteristic or set of characteristics of the hydrogel bead may bechanged by one or more conditions. A condition suitable for changing acharacteristic or set of characteristics of the hydrogel bead may be,for example, a temperature, a pH, an ion or salt concentration, apressure, chemical species, any combinations thereof, or anothercondition. For example, hydrogel bead may be exposed to a chemicalspecies that may bring about a change in one or more characteristics ofthe hydrogel bead. In some cases, a stimulus may be used to change oneor more characteristics of the hydrogel bead. For example, uponapplication of the stimulus, one or more characteristics of the hydrogelbead may be changed. The stimulus may be, for example, a thermalstimulus, a photo stimulus, a chemical stimulus, or another stimulus. Atemperature sufficient for changing one or more characteristics of thehydrogel bead may be, for example, at least about 0 degrees Celsius (°C.), 1° C., 2° C., 3° C., 4° C., 5° C., 10° C., or higher. For example,the temperature may be about 4° C. In other cases, a temperaturesufficient for changing one or more characteristics of the hydrogel beadmay be, for example, at least about 25° C., 30° C., 35° C., 37° C., 40°C., 45° C., 50° C., or higher. For example, the temperature may be about37° C. A pH sufficient for changing one or more characteristics of thehydrogel bead may be, for example, between about 5 and 8, such asbetween about 6 and 7.

In some cases, a chemical species or a chemical stimulus may be used tochange one or more characteristics of the hydrogel bead. For example, achemical species or a chemical stimulus may be used to change adimension of a hydrogel bead (e.g., a cross-section, diameter, orvolume). In some cases, a chemical species or a chemical stimulus may beused to change a dimension of a hydrogel bead (e.g., a cross-sectionaldiameter) from a first dimension to a second dimension (e.g., a secondcross-sectional dimeter), where the second dimension is reduced comparedto the first dimension. The chemical species may comprise an organicsolvent, such as an alcohol, ketone, or aldehyde. For example, thechemical species may comprise acetone, methanol, ethanol, formaldehyde,or glutaraldehyde. The chemical species may comprise a cross-linkingagent. For example, the cross-linking agent may comprise disuccinimidylsuberate (DSS), dimethylsuberimidate (DMS), formalin, anddimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP),disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidylsuccinate) (EGS), and any combinations thereof. In some cases, across-linking agent may be a photo-cleavable cross-linking agent. Insome cases, a chemical stimulus may be used to change one or morecharacteristics of the hydrogel bead (e.g., a dimension of a hydrogelbead), where the chemical stimulus comprises one or more chemicalspecies. For example, the chemical stimulus may comprise a firstchemical species and a second chemical species, where the first chemicalspecies is an organic solvent and the second chemical species is across-linking agent. In some cases, a chemical stimulus may comprise achemical species that is a fixation agent that is capable of fixing orpreserving a hydrogel bead. For example, an organic solvent such as analcohol (e.g., ethanol or methanol), ketone (e.g., acetone), or aldehyde(e.g., formaldehyde or glutaraldehyde), or any combinations thereof mayact as a fixation agent. Alternatively, or in addition, a cross-linkingagent may act as a fixation agent. In some cases, a fixation agent maycomprise disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS),formalin, and dimethyladipimidate (DMA), dithio-bis(-succinimidylpropionate) (DSP), disuccinimidyl tartrate (DST), and/or ethylene glycolbis(succinimidyl succinate) (EGS), and any combinations thereof. In somecases, a first chemical species and/or fixation agent may be provided toor brought into contact with the hydrogel bead to bring about a changein a first characteristic or set of characteristics of the hydrogelbead, and a second chemical species and/or fixation agent may beprovided to or brought into contact with the hydrogel bead to bringabout a change in a second characteristic or set of characteristics ofthe hydrogel bead. For example, a first chemical species and/or fixationagent may be provided to or brought into contact with the hydrogel beadto bring about a change in a dimension of a hydrogel bead (e.g., areduction in cross-sectional diameter), and a second chemical speciesand/or fixation agent may be provided to or brought into contact withthe hydrogel bead to bring about a change in a second characteristic orset of characteristics of the hydrogel bead (e.g., forming crosslinkswithin and/or surrounding the hydrogel bead). The first and secondchemical species and/or fixation agents may be provided to or broughtinto contact with the hydrogel bead at the same or different times.

In some embodiments, fixation may affect one or more parameters orcharacteristics of the hydrogel bead. For example, fixation may resultin shrinkage or volumetric reduction of the hydrogel bead. Fixation mayinclude dehydration of the hydrogel bead. Providing a fixation agent tothe hydrogel bead may result in a change in a dimension of the hydrogelbead. For example, providing a fixation agent to the hydrogel bead mayresult in a change in the volume or diameter of the hydrogel bead.Providing a fixation agent to the hydrogel bead may result in a changein a cross-section of the hydrogel bead (e.g., a cross-sectionaldiameter). For example, a first cross-section of the hydrogel bead priorto fixation may be different (e.g., larger) than a second cross-sectionof the hydrogel bead following fixation. In an example, an approximatelyspherical hydrogel bead may comprise a first cross section (e.g., across-sectional diameter) prior to fixation that is reduced in size to asecond cross-section following fixation. Providing a fixation agent tothe hydrogel bead may result in a second cross-section that is reducedby at least about 5% compared to the first cross-section. In some cases,the second cross-section may be reduced by at least 6%, 8%, 10%, 15%,25%, 30%, 35%, 40%, 45%, 50%, or more relative to the firstcross-section. For example, the second cross-section may be reduced byat least about 10%, 15%, 25%, or 50% relative to the firstcross-section. Fixation may also affect other features of the hydrogelbead. For example, fixation may result in a change in the porosity of amembrane or wall of a hydrogel bead, reorganization of components of thehydrogel bead, a change in hydrogel bead fluidity or rigidity, or otherchanges. In an example, a first fixation agent that is an organicsolvent is provided to the hydrogel bead to change a firstcharacteristic (e.g., hydrogel bead volume) and a second fixation agentthat is a cross-linking agent is provided to the hydrogel bead to changea second characteristic (e.g., hydrogel bead fluidity or rigidity). Thefirst fixation agent may be provided to the hydrogel bead before thesecond fixation agent.

In some instances, an approximately spherical hydrogel bead may comprisea first diameter prior to fixation (e.g., by an organic solvent) that isreduced in volume compared to a second diameter following fixation whenmaintained in a non-aqueous environment. Following fixation andreduction in volume to said second diameter, when maintained in anaqueous environment, the hydrogel bead may increase in volume to have adiameter substantially similar to the first diameter. In some cases, anapproximately spherical hydrogel bead may include a first diameter priorto fixation (e.g., by an organic solvent) that is reduced in volumecompared to a second diameter following fixation. Following fixation andreduction in volume to said second diameter, the hydrogel bead may becross-linked by a second fixative, wherein the second diameter issubstantially maintained in an aqueous environment followingcross-linking by the second fixative.

A change to a characteristic or set of characteristics of the hydrogelbead may be reversible or irreversible. In some cases, a change to acharacteristic or set of characteristics of the hydrogel bead may beirreversible, such that the change cannot be readily undone. Forexample, the volume, morphology, or other feature of the hydrogel beadmay be altered in a way that cannot be readily reversed. In an example,the change from a first cross-section of the hydrogel bead to a secondcross-section of the hydrogel bead is irreversible. In some cases, anirreversible change may be at least partially reversed upon theapplication of appropriate conditions and/or over a period of time. Inother cases, a change to a characteristic or set of characteristics ofthe hydrogel bead may be reversible. For example, the volume of ahydrogel bead may be reduced upon being subjected to a first conditionor set of conditions, and the volume of a hydrogel bead may be increasedto approximately the original volume upon being subjected to a secondcondition or set of conditions. Thus, the change from a firstcross-section of the hydrogel bead to the second cross-section may bereversible. A reversible change (e.g., a reversible volume reduction)may be useful in, for example, providing a hydrogel bead of a givenvolume to a given location, such as a partition. In some cases, a changeto a characteristic or set of characteristics of the hydrogel bead maybe only partially reversible. For example, the volume of a hydrogel beadmay be reduced (e.g., by dehydration), and the reduction in hydrogelbead volume may be accompanied by reorganization of components withinthe hydrogel bead. Upon reversal of the volume of the hydrogel bead(e.g., by rehydration), the arrangement of one or more components maynot revert to the original arrangement of the hydrogel bead prior to thevolume reduction. A change to a characteristic or set of characteristicsof the hydrogel bead, such as a cross-section of the hydrogel bead, maybe reversible upon application of a stimulus. The stimulus may be, forexample, a thermal stimulus, a photo stimulus, or a chemical stimulus.In some cases, the stimulus may comprise a change in pH and/orapplication of a reducing agent such as dithiothreitol. Application ofthe stimulus may reverse, wholly or in part, a change from, for example,a first cross-section to a second cross-section.

In some embodiments, a plurality of hydrogel beads can be shrunkenhydrogel beads generated by removing water from a plurality of firsthydrogel beads. In some embodiments, the plurality of shrunken hydrogelbeads has an average diameter no larger than the diameter of a cell(e.g., a mammalian cell, a plant cell, or a fungal cell). In someembodiments, each member of the plurality of shrunken hydrogel beads hasa diameter no larger than the diameter of a cell (e.g., a mammaliancell, a plant cell, or a fungal cell).

In some embodiments, bead arrays having a plurality of hydrogel beadsdisposed on a substrate are generated by patterning or self-assembly oflarger gel beads, after which the array of larger gel beads is shrunken(e.g., by any of the variety of methods provided herein). In someembodiments, the larger gel beads are not small enough for single-cellresolution, while the shrunken gel beads are small enough forsingle-cell resolution. In some embodiments, bead arrays having aplurality of hydrogel beads disposed on a substrate are generated bypatterning or self-assembly of shrunken gel beads that have previouslybeen generated by shrinking larger gel beads (e.g., by any of thevariety of methods provided herein). Beads can be spatially confined byany of a variety of methods, including without limitation, reversible orirreversible crosslinking.

In some embodiments, bead arrays include spatially-confined gel beadswith high aspect ratios (e.g., pillared arrays). For example, beadarrays having a plurality of hydrogel beads disposed on a substrate canbe generated by any of the variety of methods described herein (e.g., bypatterning or self-assembly of shrunken gel beads or by patterning orself-assembly of larger gel beads followed by shrinking), after whichthe high-density bead array is expanded (or re-expanded). Whenexpanding, spatial constraints direct the beads to expand primarily inthe Z dimension (away from the substrate), resulting in pillar arrays.In some embodiments, the gel beads of the pillar arrays have high aspectratios. In some embodiments, aspect ratio of the expanded plurality ofspatially-confined shrunken hydrogel beads is at least 2. In otherembodiments, the aspect ratio of the expanded plurality ofspatially-confined shrunken hydrogel beads is at least 3. In someembodiments, the plurality of spatially-confined shrunken hydrogel beadshas an average aspect ratio of at least 4, 5, 6, 7, 8 or more.

In some embodiments, the method for the removal of water from a hydrogelis the same for each hydrogel (e.g., the first hydrogel, the secondhydrogel, or the third hydrogel). In some embodiments, the method forthe removal of water from one hydrogel (e.g., the first hydrogel) isdifferent from the method for the removal of water for at least oneother hydrogel (e.g., a second hydrogel, a third hydrogel, or a fourthhydrogel). For example, the method for the removal of water from onehydrogel can be different from the method for the removal of water forthe other hydrogels (e.g., a second hydrogel, a third hydrogel, or afourth hydrogel). In some embodiments, the method for the removal ofwater is different for each hydrogel (e.g., the first hydrogel, thesecond hydrogel, the third hydrogel, and the fourth hydrogel).

In some embodiments, the shrunken hydrogel is at least about 2-foldsmaller in a linear dimension (e.g., along one axis) than the pre-shrunkhydrogel. For example, at least about 2.5, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, or more fold smaller in alinear dimension than the pre-shrunk hydrogel.

In some embodiments, the size of the hydrogel is reduced along more thanone axes, e.g., along 2 or 3 axes. In some embodiments, the reduction inthe volume of the hydrogel is isometric.

In some embodiments, the volume of each hydrogel (e.g., a firsthydrogel, a second hydrogel, a third hydrogel, or a fourth hydrogel) isthe same. In some embodiments, the volume of at least one hydrogel isdifferent. For example, in some embodiments, one hydrogel is differentin volume from the other hydrogels (e.g., a second hydrogel, a thirdhydrogel, or a fourth hydrogel). In some embodiments, every hydrogel isdifferent in volume from every other hydrogel.

In some embodiments, members of the plurality of features arecross-linked to a hydrogel (e.g., a first hydrogel, a second hydrogel, athird hydrogel, or a fourth hydrogel).

In one embodiment, features of an array can be copied into a hydrogel,and the volume of the hydrogel is reduced by removing water. These stepscan be performed multiple times. For example, a method for preparing ahigh-density spatially-barcoded flexible array can include copying aplurality of spatially-barcoded features from an array into a firsthydrogel, wherein the first hydrogel is in contact with the array;reducing the volume of the first hydrogel including the copied featuresby removing water, forming a first shrunken hydrogel including thecopied features; copying the features in the first shrunken hydrogelinto a second hydrogel, where the second hydrogel is in contact with thefirst hydrogel; and reducing the volume of the second hydrogel includingthe copied features by removing water, forming a second shrunkenhydrogel including the copied features, thus generating a high-densityspatially-barcoded array. In some instances, the array includes one ormore pluralities of first oligonucleotides and the first hydrogelincludes one or more pluralities of second oligonucleotides. Uponcontacting the hydrogel with the array, members of the one or morepluralities of the first oligonucleotides can be attached to members ofthe one or more pluralities of second oligonucleotides. The array caninclude more species of first oligonucleotides than the number ofspecies of the second oligonucleotides in the hydrogel such that firstoligonucleotides comprising the same sequence can be coupled to secondoligonucleotides comprising different sequences. The diversity of theoligonucleotides (e.g., spatial barcodes) in the first hydrogel canthereby be increased. The process of copying spatially-barcoded featuresfrom an array to a first hydrogel, removing water from the firsthydrogel to form a first shrunken hydrogel, and copyingspatially-barcoded features from the first shrunken hydrogel to one ormore subsequent hydrogels can be performed multiple times (e.g., 2, 3,4, 5, 6, 7, 8, 9, or 10 times). The result is a high-density flexiblearray including spatially-barcoded features.

In some embodiments, copying members of the plurality of features froman array includes copy by PCR. In some embodiments, the hydrogel (e.g.,a first hydrogel, a second hydrogel, a third hydrogel, and/or a fourthhydrogel) comprises PCR reagents as described herein. In someembodiments, members of the plurality of features are copied usingreplica plating techniques (see, e.g., Mitra and Church, Nucleic AcidsRes. 1999 Dec. 15; 27(24):e34, which is incorporated by reference hereinin its entirety). In some embodiments, after copying a plurality offeatures from an array into a first hydrogel, the features of the arrayare amplified in the first hydrogel (e.g., clonal amplification). Insome embodiments, members of the plurality of features are copied intothe first hydrogel such that the pattern of the plurality of features ofthe first hydrogel is the same or substantially similar (e.g., at least80%) to the pattern of the plurality of features of the array.

In some embodiments, one or more pluralities of features of the arrayare partitioned. For example, each partition can comprise a plurality offeatures different from the plurality of features of other partitions.For example, the members of the plurality of features are partitionedsimilar to the partitions of the plurality of features of the array. Insome embodiments, the features of the array are copied into the firsthydrogel, such that the volume or diameter of the pre-shrunk firsthydrogel features are similar to the volume or diameter of the arrayfeatures.

In some embodiments, the volume of a hydrogel comprising copied featuresis reduced, thus increasing the density of the copied features. In someembodiments, the copied features within a hydrogel further increases indensity with each subsequent hydrogel copy and shrinking. For example,the density of the copied features of a second shrunken hydrogel ishigher than the density of the copied features of a first shrunkenhydrogel. Similarly, the density of the copied features of a thirdshrunken hydrogel is higher than the density of the copied features of asecond shrunken hydrogel. Similarly, the density of the copied featuresof a fourth shrunken hydrogel is higher than the density of the copiedfeatures of a third shrunken hydrogel. In some embodiments, the volumeof a partition of members of the plurality of features in a hydrogel isreduced when the volume of the hydrogel is reduced.

In some embodiments of the methods described herein, an array comprisesshrunken gel features (e.g., beads). In some embodiments, the methodsdescribed herein generate shrunken gel bead arrays. In some embodiments,the shrunken gel beads of the array are shrunken hydrogel beads.

A “shrunken array” includes a plurality of spatially-barcoded featuresattached to, or embedded in, a substrate that have been reduced involume (e.g., reduction in diameter or volume). A biological sample canbe contacted with a shrunken array and further contacted with a solutioncapable of rehydrating the shrunken array. In some embodiments, analytetransfer and capture is driven by molecular diffusion. The process ofrehydrating the shrunken array by providing a permeabilization solutionor tissue stain to the sample can promote analytes (e.g., transcripts)present in the biological sample towards the spatially-barcodedfeatures, thereby improving capture efficiency of the analytes. See,e.g., J. Vlassakis, A. E. Herr. “Effect of Polymer Hydration State onIn-Gel Immunoassays.” Anal. Chem. 2015, 87(21):11030-8, hereinincorporated by reference in its entirety.

A shrunken array can be generated with features (e.g., beads) containingspatial barcodes from an existing array. For example, an array (e.g.,hydrogel bead array) described and prepared by any method herein can becontacted with reagents capable of dehydrating (e.g., removing water)the features (e.g., beads) to generate a shrunken array (e.g., ashrunken bead array). Methods of dehydrating features (e.g., beads) areknown in the art. Any suitable method of dehydration (e.g., removingwater) can be used.

In some embodiments, the dehydrated features (e.g., beads) can create ashrunken array (e.g., shrunken bead array or shrunken hydrogel array)where the average diameter of the dehydrated features (e.g., beads) canbe smaller than the average diameter of the features prior todehydration. In some embodiments, the dehydrated features (e.g., beads)can have an average diameter at least two-fold smaller than the averagediameter of the features prior to dehydration. In some embodiments, thedehydrated features (e.g., beads) can have an average diameter at leastthree-fold smaller than the average diameter of the features prior todehydration. In some embodiments, the dehydrated features (e.g., beads)can have an average diameter at least four-fold or smaller than theaverage diameter of the features (e.g., beads) prior to dehydration.

After generating a shrunken array, a biological sample (e.g., tissuesample) can be contacted with the shrunken array (e.g., shrunken beadarray). A rehydrating solution can be provided to the biological sampleand the shrunken array by any suitable method (e.g., by pipetting). Therehydrating solution can contain reagents to rehydrate (e.g., water orbuffers) the features (e.g., beads) of the shrunken array. In someembodiments, the rehydrating solution can be applied to the entirebiological sample. In some embodiments, the rehydrating solution can beselectively applied (e.g., to a region of interest). In someembodiments, absorbing water from the rehydrating solution can increasethe diameter of at least one feature (e.g., bead) in the shrunken array.In some embodiments, the rehydrating solution can increase the diameterof at least one feature (e.g., bead) by at least two-fold. In someembodiments, the rehydrating solution can increase the diameter of atleast one feature (e.g., bead) by at least three-fold. In someembodiments, the rehydrating solution can increase the diameter of atleast one feature (e.g., bead) by at least four-fold. In someembodiments, the rehydrating solution can increase the diameter of atleast one feature (e.g., bead) by at least five-fold or more.

In some embodiments, the rehydrating solution can containpermeabilization reagents. The biological sample can be permeabilizedusing permeabilization reagents and techniques known in the art orotherwise described herein. Biological samples from different sources(e.g., brain, liver, ovaries, kidney, breast, colon, etc.) can requiredifferent permeabilization treatments. For example, permeabilizing thebiological sample (e.g., using a protease) can facilitate the migrationof analytes to the substrate surface (e.g., spatially-barcodedfeatures). In some embodiments, the permeabilization reagents can be adetergent (e.g., saponin, Triton X100™, Tween-20™). In some embodiments,an organic solvent (e.g., methanol, acetone) can permeabilize cells ofthe biological sample. In some embodiments, an enzyme (e.g., trypsin)can permeabilize the biological sample. In another embodiment, an enzyme(e.g., collagenase) can permeabilize the biological sample.

In some embodiments the solution can permeabilize the biological sampleand rehydrate the features (e.g., beads) of the shrunken array (e.g.,shrunken hydrogel). In some embodiments, the rehydrating solution canstain the biological sample and rehydrate the features of the shrunkenarray (e.g., beads).

In some embodiments, the rehydrating solution (e.g., permeabilization orstain solution) can diffuse through the biological sample. In someembodiments, the rehydrating solution can reduce diffusion of analytesaway from the substrate. In some embodiments, while diffusing throughthe biological sample, the rehydrating solution can migrate analytestoward the substrate surface and improve the efficiency of analytecapture.

(vii) Microcapillary Arrays

A “microcapillary array” is an arrayed series of features that arepartitioned by microcapillaries. A “microcapillary channel” is anindividual partition created by the microcapillaries. For example,microcapillary channels can be fluidically isolated from othermicrocapillary channels, such that fluid or other contents in onemicrocapillary channel in the array are separated from fluid or othercontents in a neighboring microcapillary channel in the array. Thedensity and order of the microcapillary channels can be any suitabledensity or order of discrete sites.

In some embodiments, microcapillary arrays are treated to generateconditions that facilitate loading.

In some embodiments, when random distribution is used to distributefeatures, empirical testing can be performed to generateloading/distribution conditions that facilitate a single feature permicrocapillary.

In some embodiments, the microcapillary array is placed in contact witha sample (e.g., on top or below) so that microcapillaries containing afeature (e.g., a bead, which can include a capture probe) are in contactwith the biological sample.

In some embodiments, a biological sample is partitioned by contacting amicrocapillary array to the biological sample, thereby creatingmicrocapillary channels including a bead and a portion of the biologicalsample.

In some embodiments, reagents are added to the microcapillary array.

In some embodiments, a reagent solution is removed from eachmicrocapillary channel following an incubation for an amount of time andat a certain temperature or range of temperatures, e.g., following ahybridization or an amplification reaction. Hydrogel/Well Arrays

In some embodiments are methods for generating patterned hydrogel arraysusing wells (e.g., a nanowell or microwell array).

In some embodiments, the method includes providing shrunken (e.g.,dehydrated) hydrogel features (e.g., beads) to a well array.

In some embodiments, the method includes rehydrating (e.g., addingwater) the shrunken hydrogel features, wherein the shrunken hydrogelfeatures are located in the wells. Bead Tethering

“Bead tethering” can refer to an arrangement of beads, wherein thearrangement may or may not form an array. The tethered beads can becontacted with a sample and processed according to methods describedherein. Further, contacting a biological sample with a single bead orbeads tethered together in various arrangements can allow for moreprecise spatial detection of analytes, e.g., a region of interest.Methods for tethering beads together are known in the art. Somesuitable, non-limiting, methods of tethering beads together can be,e.g., chemical linkers, proximity ligation, or any other methoddescribed herein. In some embodiments, beads can be tethered togetherindependent of a substrate. In some embodiments, beads can be tetheredin various arrangements on an existing substrate. In some embodiments, asubstrate (e.g., a hydrogel) can be formed around existing tetheredbeads. In some embodiments, the beads or bead arrangement can contact aportion of the biological sample. In some embodiments, the bead or beadarrangement can contact a region of interest. In some embodiments, thebeads or bead arrangement can contact the entire biological sample. Insome embodiments, the beads or bead arrangements are contacted to randompositions on the biological sample. In some embodiments, the beads arecontacted to according to a specific pattern on the biological sample.

Beads can be tethered together in various arrangements. In someembodiments, a single (e.g., one) bead can be contacted with abiological sample. In some embodiments, two or more beads can betethered (e.g., connected to each other), in various arrangements. Forexample, in a non-limiting way, beads can be tethered together to form acluster, a row, or arranged on a mesh (e.g., a net). In someembodiments, at least three beads can be tethered together in atwo-dimensional (2D) array (e.g., a cluster). In some embodiments, atleast two beads can be tethered together in a one-dimensional (1D) array(e.g., a row). In such embodiments, the beads are arranged in suchfashion that the beads can contact each other directly. In someembodiments, at least two beads can be tethered together in a stringarrangement. In such embodiments, the beads are arranged in such fashionthat the beads can contact each other indirectly (e.g., beads areconnected via linker). In some embodiments, at least two beads can betethered together in a mesh arrangement (e.g., net). In someembodiments, beads tethered together in a 2D array, a 1D array, thebeads on a string arrangement, and the beads on the mesh arrangement canbe used in any combination with each other on the biological sample.

In some embodiments, the tethered beads can have capture probescomprising spatial barcodes, functional domains, unique molecularidentifiers, cleavage domains, and capture domains, or combinationsthereof. In some embodiments, each bead can be associated with a uniquespatial barcode. In some embodiments, the spatial barcode is known priorto contacting the bead or bead arrangement to the biological sample. Insome embodiments, the spatial barcode is not known prior to contactingthe bead or bead arrangement to the biological sample. The identity ofeach bead (e.g., spatial barcode) in the array can be deconvolved, forexample, by direct optical sequencing, as discussed herein.

(viii) Printing Arrays in Liquid

In some embodiments, an array can be printed in liquid. The resolutionof conventionally-printed arrays can be limited, due to the diffusion ofprinted solutions. Printing the array in a highly viscous liquid canincrease resolution by preventing the diffusion of the printed solution.Thus, disclosed herein are various methods and materials for attachingand/or introducing a capture probe (e.g., a nucleic acid capture probe)having a barcode (e.g., a spatial barcode) to a substrate (e.g., aslide), wherein the attaching (e.g., printing) is performed in liquid.

In some aspects, capture probes are printed on a substrate (e.g., aslide or bead). In some aspects, the substrate is a slide. In someaspects, the substrate is a 96-well microtiter plate. In some aspects,methods provided herein can also be applied to other substrates commonlyused for nucleic acid analyses, including but not limited to beads,particles, membranes, filters, dipsticks, slides, plates, andmicrochips. In some aspects, such substrates may be composed of a numberof materials known to be compatible with nucleic acid analysis,including but not limited to agarose, styrene, nylon, glass, andsilicon.

Array/Feature Preservation

In some embodiments, the biological sample can be preserved aftercompletion of an assay with a feature or arrangement of features foradditional rounds of spatial detection of analytes. In some embodiments,the biological sample, features, array, or any combination thereof canbe preserved after the spatial profiling. In some embodiments, thebiological sample, features, array, or combinations thereof can beprotected from dehydration (e.g., drying, desiccation). In someembodiments, the biological sample, features, array, or combinationsthereof, can be protected from evaporation. Methods of preserving and/orprotecting biological samples, features, or arrays are known in the art.For example, in a non-limiting way, the biological sample, features,array, or combinations thereof can be covered by a reversible sealingagent. Any suitable reversible sealing agent can be used. Methods ofreversible sealing are known in the art (See, e.g., WO 2019/104337,which is incorporated herein by reference). In a non-limiting way,suitable reversible sealing agents can include non-porous materials,membranes, lids, or oils (e.g., silicone oil, mineral oil). In furthernon-limiting examples, the biological sample, features, array, orcombinations thereof can be preserved in an environmental chamber (e.g.,hermetically sealed) and removed for additional rounds of spatialanalysis at a later time.

(e) Analyte Capture

In this section, general aspects of methods and systems for capturinganalytes are described. Individual method steps and system features canbe present in combination in many different embodiments; the specificcombinations described herein do not in any way limit other combinationsof steps or features.

(i) Conditions for Capture

Generally, analytes can be captured when contacting a biological samplewith a substrate including capture probes (e.g., substrate with captureprobes embedded, spotted, printed on the substrate or a substrate withfeatures (e.g., beads, wells) comprising capture probes).

As used herein, “contact,” “contacted,” and/or “contacting,” abiological sample with a substrate refers to any contact (e.g., director indirect) such that capture probes can interact (e.g., capture) withanalytes from the biological sample. For example, a substrate may benear or adjacent to the biological sample without direct physicalcontact, yet capable of capturing analytes from the biological sample.In some embodiments a biological sample is in direct physical contactwith a substrate. In some embodiments, a biological sample is inindirect physical contact with a substrate. For example, a liquid layermay be between the biological sample and the substrate. In someembodiments, analytes diffuse through a liquid layer. In someembodiments capture probes diffuse through a liquid layer. In someembodiments reagents may be delivered via a liquid layer between abiological sample and a substrate. In some embodiments, indirectphysical contact may include a second substrate (e.g., a hydrogel, afilm, a porous membrane) between the biological sample and the firstsubstrate comprising capture probes. In some embodiments, reagents maybe delivered by a second substrate to a biological sample.

In some embodiments, a cell immobilization agent can be used to contacta biological sample with a substrate (e.g., by immobilizingnon-aggregated or disaggregated sample on a spatially-barcoded arrayprior to analyte capture). A “cell immobilization agent” as used hereincan refer to an agent (e.g., an antibody), attached to a substrate,which can bind to a cell surface marker. Non-limiting examples of a cellsurface marker include CD45, CD3, CD4, CD8, CD56, CD19, CD20, CD11c,CD14, CD33, CD66b, CD34, CD41, CD61, CD235a, CD146, and epithelialcellular adhesion molecule (EpCAM). A cell immobilization agent caninclude any probe or component that can bind to (e.g., immobilize) acell or tissue when on a substrate. A cell immobilization agent attachedto the surface of a substrate can be used to bind a cell that has a cellsurface maker. The cell surface marker can be a ubiquitous cell surfacemarker, wherein the purpose of the cell immobilization agent is tocapture a high percentage of cells within the sample. The cell surfacemarker can be a specific, or more rarely expressed, cell surface marker,wherein the purpose of the cell immobilization agent is to capture aspecific cell population expressing the target cell surface marker.Accordingly, a cell immobilization agent can be used to selectivelycapture a cell expressing the target cell surface marker from apopulation of cells that do not have the same cell surface marker.

Capture probes on a substrate (or on a feature on the substrate) mayinteract with released analytes through a capture domain, describedelsewhere, to capture analytes. In some embodiments, certain steps areperformed to enhance the transfer or capture of analytes to the captureprobes of the array. Examples of such modifications include, but are notlimited to, adjusting conditions for contacting the substrate with abiological sample (e.g., time, temperature, orientation, pH levels,pre-treating of biological samples, etc.), using force to transportanalytes (e.g., electrophoretic, centrifugal, mechanical, etc.),performing amplification reactions to increase the amount of biologicalanalytes (e.g., PCR amplification, in situ amplification, clonalamplification), and/or using labeled probes for detecting of ampliconsand barcodes.

In some embodiments, an array is adapted in order to facilitatebiological analyte migration. Non-limiting examples of adapting an arrayto facilitate biological analyte migration include arrays withsubstrates containing nanopores, nanowells, and/or microfluidicchannels; arrays with porous membranes; and arrays with substrates thatare made of hydrogel. In some cases, the array substrate is liquidpermeable. In some cases, the array is a coverslip or slide thatincludes nanowells or patterning, (e.g., via fabrication). In some caseswhere the substrate includes nanopores, nanowells, and/or microfluidicchannels, these structures can facilitate exposure of the biologicalsample to reagents (e.g., reagents for permeabilization, biologicalanalyte capture, and/or a nucleic acid extension reaction), therebyincreasing analyte capture efficiency as compared to a substrate lackingsuch characteristics.

In some embodiments, analyte capture is facilitated by treating abiological sample with permeabilization reagents. If a biological sampleis not permeabilized sufficiently, the amount of analyte captured on asubstrate can be too low to enable adequate analysis. Conversely, if abiological sample is too permeable, an analyte can diffuse away from itsorigin in the biological sample, such that the relative spatialrelationship of the analytes within the biological sample is lost.Hence, a balance between permeabilizing the biological sample enough toobtain good signal intensity while still maintaining the spatialresolution of the analyte distribution in the biological sample isdesired. Methods of preparing biological samples to facilitate analytecapture are known in the art and can be modified depending on thebiological sample and how the biological sample is prepared (e.g., freshfrozen, FFPE, etc.).

(ii) Substrate Holder

Described herein are methods in which an array with capture probeslocated on a substrate and a biological sample located on a differentsubstrate, are contacted such that the array is in contact with thebiological sample (e.g., the substrates are sandwiched together). Insome embodiments, the array and the biological sample can be contacted(e.g., sandwiched), without the aid of a substrate holder. In someembodiments, the array and biological sample substrates can be placed ina substrate holder (e.g., an array alignment device) designed to alignthe biological sample and the array. For example, the substrate holdercan have placeholders for two substrates. In some embodiments, an arrayincluding capture probes can be positioned on one side of the substrateholder (e.g., in a first substrate placeholder). In some embodiments, abiological sample can be placed on the adjacent side of the substrateholder in a second placeholder. In some embodiments, a hinge can belocated between the two substrate placeholders that allows the substrateholder to close, e.g., make a sandwich between the two substrateplaceholders. In some embodiments, when the substrate holder is closedthe biological sample and the array with capture probes are contactedwith one another under conditions sufficient to allow analytes presentin the biological sample to interact with the capture probes of thearray. For example, dried permeabilization reagents can be placed on thebiological sample and rehydrated. A permeabilization solution can beflowed through the substrate holder to permeabilize the biologicalsample and allow analytes in the biological sample to interact with thecapture probes. Additionally, the temperature of the substrates orpermeabilization solution can be used to initiate or control the rate ofpermeabilization. For example, the substrate including the array, thesubstrate including the biological sample, or both substrates can beheld at a low temperature to slow diffusion and permeabilizationefficiency. Once sandwiched, in some embodiments, the substrates can beheated to initiate permeabilization and/or increase diffusionefficiency. Transcripts that are released from the permeabilized tissuecan diffuse to the array and be captured by the capture probes. Thesandwich can be opened, and cDNA synthesis can be performed on thearray.

Any of the variety of combinations described herein where a sandwichincluding an array with capture probes and a biological sample on twodifferent substrates can be placed in a substrate holder designed toalign the biological sample and the array. For example, the substrateholder can have placeholders for two substrates. In some embodiments, anarray including capture probes can be positioned on one side of thesubstrate holder (e.g., in a first substrate placeholder). In someembodiments, a biological sample can be placed on the adjacent side ofthe substrate holder in a second placeholder. In some embodiments, inbetween the two substrate placeholders can be a hinge that allows thesubstrate holder to close, e.g., make a sandwich between the twosubstrate placeholders. In some embodiments, when the substrate holderis closed the biological sample and the array with capture probes can becontacted with one another under conditions sufficient to allow analytespresent in the biological sample to interact with the capture probes ofthe array for spatial analysis by any method described herein. Forexample, dried permeabilization reagents can be placed on the biologicalsample and rehydrated. Additionally, a permeabilization solution can beflowed through the substrate holder to permeabilize the biologicalsample and allow analytes in the biological sample to interact with thecapture probes.

In some embodiments, a flexible array described herein can be placed inthe substrate holder, and sandwiched with a biological sample. In someembodiments, the flexible array can include spatially-barcodedcross-linked features. In some embodiments, the flexible array can bepresoaked in permeabilization reagents before being placed into thesubstrate holder. In some embodiments, the flexible array can be soakedin permeabilization reagents after being placed in the substrate holder.In some embodiments, the substrate holder including a biological samplein one placeholder and a flexible array can be closed (e.g., form asandwich) such that the permeabilization reagents allow analytes presentin the biological sample to interact with capture probes of the flexiblearray (e.g., capture probes on the spatially-barcoded features).

In some embodiments, the substrate holder can be heated or cooled toregulate permeabilization and/or diffusion efficiency.

(iii) Passive Capture Methods

In some embodiments, analytes can be migrated from a sample to asubstrate. Methods for facilitating migration can be passive (e.g.,diffusion) and/or active (e.g., electrophoretic migration of nucleicacids). Non-limiting examples of passive migration can include simplediffusion and osmotic pressure created by the rehydration of dehydratedobjects.

Passive migration by diffusion uses concentration gradients. Diffusionis movement of untethered objects toward equilibrium. Therefore, whenthere is a region of high object concentration and a region of lowobject concentration, the object (e.g., a capture probe, an analyte,etc.) moves to an area of lower concentration. In some embodiments,untethered analytes move down a concentration gradient.

In some embodiments, different reagents may be added to the biologicalsample, such that the biological sample is rehydrated while improvingcapture of analytes. In some embodiments, the biological sample can becontacted with a shrunken array as described herein. In someembodiments, the biological sample and/or the shrunken array can berehydrated with permeabilization reagents. In some embodiments, thebiological sample and/or the shrunken array can be rehydrated with astaining solution (e.g., hematoxylin and eosin stain).

(iv) Diffusion-Resistant Media/Lids

To increase efficiency by encouraging analyte diffusion toward thespatially-barcoded capture probes, a diffusion-resistant medium can beused. In general, molecular diffusion of biological analytes occurs inall directions, including toward the capture probes (i.e., toward thespatially-barcoded array), and away from the capture probes (i.e., intothe bulk solution). Increasing diffusion toward the spatially-barcodedarray reduces analyte diffusion away from the spatially-barcoded arrayand increases the capturing efficiency of the capture probes.

In some embodiments, a biological sample is placed on the top of aspatially-barcoded substrate and a diffusion-resistant medium is placedon top of the biological sample. For example, the diffusion-resistantmedium can be placed onto an array that has been placed in contact witha biological sample. In some embodiments, the diffusion-resistant mediumand spatially-barcoded array are the same component. For example, thediffusion-resistant medium can contain spatially-barcoded capture probeswithin or on the diffusion-resistant medium (e.g., coverslip, slide,hydrogel, or membrane). In some embodiments, a sample is placed on asubstrate and a diffusion-resistant medium is placed on top of thebiological sample. Additionally, a spatially-barcoded capture probearray can be placed in close proximity over a diffusion-resistantmedium. For example, a diffusion-resistant medium may be sandwichedbetween a spatially-barcoded array and a sample on a substrate. In someembodiments, a diffusion-resistant medium is disposed or spotted onto asample. In other embodiments, a diffusion-resistant medium is placed inclose proximity to a sample.

In general, a diffusion-resistant medium can be any material known tolimit diffusivity of biological analytes. For example, adiffusion-resistant medium can be a solid lid (e.g., coverslip or glassslide). In some embodiments, a diffusion-resistant medium may be made ofglass, silicon, paper, hydrogel polymer monoliths, or other material. Insome embodiments, the glass side can be an acrylated glass slide. Insome embodiments, the diffusion-resistant medium is a porous membrane.In some embodiments, the material may be naturally porous. In someembodiments, the material may have pores or wells etched into solidmaterial. In some embodiments, the pore volume can be manipulated tominimize loss of target analytes. In some embodiments, the membranechemistry can be manipulated to minimize loss of target analytes. Insome embodiments, the diffusion-resistant medium (e.g., hydrogel) iscovalently attached to a substrate (e.g., glass slide). In someembodiments, a diffusion-resistant medium can be any material known tolimit diffusivity of poly(A) transcripts. In some embodiments, adiffusion-resistant medium can be any material known to limit thediffusivity of proteins. In some embodiments, a diffusion-resistantmedium can be any material know to limit the diffusivity ofmacromolecular constituents.

In some embodiments, a diffusion-resistant medium includes one or morediffusion-resistant media. For example, one or more diffusion-resistantmedia can be combined in a variety of ways prior to placing the media incontact with a biological sample including, without limitation, coating,layering, or spotting. As another example, a hydrogel can be placed ontoa biological sample followed by placement of a lid (e.g., glass slide)on top of the hydrogel.

In some embodiments, a force (e.g., hydrodynamic pressure, ultrasonicvibration, solute contrasts, microwave radiation, vascular circulation,or other electrical, mechanical, magnetic, centrifugal, and/or thermalforces) is applied to control diffusion and enhance analyte capture. Insome embodiments, one or more forces and one or more diffusion-resistantmedia are used to control diffusion and enhance capture. For example, acentrifugal force and a glass slide can used contemporaneously. Any of avariety of combinations of a force and a diffusion-resistant medium canbe used to control or mitigate diffusion and enhance analyte capture.

In some embodiments, a diffusion-resistant medium, along with thespatially-barcoded array and sample, is submerged in a bulk solution. Insome embodiments, a bulk solution includes permeabilization reagents. Insome embodiments, a diffusion-resistant medium includes at least onepermeabilization reagent. In some embodiments, a diffusion-resistantmedium (i.e. hydrogel) is soaked in permeabilization reagents beforecontacting the diffusion-resistant medium to the sample. In someembodiments, a diffusion-resistant medium can include wells (e.g.,micro-, nano-, or picowells) containing a permeabilization buffer orreagents. In some embodiments, a diffusion-resistant medium can includepermeabilization reagents. In some embodiments, a diffusion-resistantmedium can contain dried reagents or monomers to deliverpermeabilization reagents when the diffusion-resistant medium is appliedto a biological sample. In some embodiments, a diffusion-resistantmedium is added to the spatially-barcoded array and sample assemblybefore the assembly is submerged in a bulk solution. In someembodiments, a diffusion-resistant medium is added to thespatially-barcoded array and sample assembly after the sample has beenexposed to permeabilization reagents. In some embodiments,permeabilization reagents are flowed through a microfluidic chamber orchannel over the diffusion-resistant medium. In some embodiments, theflow controls the sample's access to the permeabilization reagents. Insome embodiments, target analytes diffuse out of the sample and toward abulk solution and get embedded in a spatially-barcoded captureprobe-embedded diffusion-resistant medium. In some embodiments, a freesolution is sandwiched between the biological sample and adiffusion-resistant medium.

FIG. 13 is an illustration of an exemplary use of a diffusion-resistantmedium. A diffusion-resistant medium/lid 1302 can be contacted with asample 1303. In FIG. 13, a glass slide 1304 is populated withspatially-barcoded capture probes 1306, and the sample 1303, 1305 iscontacted with the array 1304, 1306. A diffusion-resistant medium/lid1302 can be applied to the sample 1303, wherein the sample 1303 issandwiched between a diffusion-resistant medium 1302 and a capture probecoated slide 1304. When a permeabilization solution 1301 is applied tothe sample, using the diffusion-resistant medium/lid 1302 directsmigration of the analytes 1305 toward the capture probes 1306 byreducing diffusion of the analytes out into the medium. Alternatively,the diffusion resistant medium/lid may contain permeabilizationreagents.

(v) Active Capture Methods

In some of the methods described herein, an analyte in a biologicalsample (e.g., in a cell or tissue section) can be transported (e.g.,passively or actively) to a capture probe (e.g., a capture probe affixedto a substrate (e.g., a substrate or bead)).

For example, analytes can be transported to a capture probe (e.g., animmobilized capture probe) using an electric field (e.g., usingelectrophoresis), pressure, fluid flow, gravity, temperature, and/or amagnetic field. For example, analytes can be transported through, e.g.,a gel (e.g., hydrogel), a fluid, or a permeabilized cell, to a captureprobe (e.g., an immobilized capture probe) using a pressure gradient, achemical concentration gradient, a temperature gradient, and/or a pHgradient. For example, analytes can be transported through a gel (e.g.,hydrogel), a fluid, or a permeabilized cell, to a capture probe (e.g.,an immobilized capture probe).

In some examples, an electrophoretic field can be applied to analytes tofacilitate migration of analytes towards a capture probe. In someexamples, a sample containing analytes contacts a substrate havingcapture probes fixed on the substrate (e.g., a slide, cover slip, orbead), and an electric current is applied to promote the directionalmigration of charged analytes towards capture probes on a substrate. Anelectrophoresis assembly (e.g., an electrophoretic chamber), where abiological sample is in contact with a cathode and capture probes (e.g.,capture probes fixed on a substrate), and where the capture probes arein contact with the biological sample and an anode, can be used to applythe current.

In some embodiments, methods utilizing an active capture method canemploy a conductive substrate (e.g., any of the conductive substratesdescribed herein). In some embodiments, a conductive substrate includespaper, a hydrogel film, or a glass slide having a conductive coating. Insome embodiments, a conductive substrate (e.g., any of the conductivesubstrates described herein) includes one or more capture probes.

FIGS. 24A and 24B show different example analytical workflows of activecapture methods using an electric field (e.g., using electrophoresis).In some examples, a biological sample 2402 (e.g., a tissue sample) canbe in contact with a first substrate 2404. In some embodiments, firstsubstrate 2404 can have one or more coatings (e.g., any of theconductive substrates described herein) on its surface. Non-limitingexamples of coatings include, nucleic acids (e.g., RNA) and conductiveoxides (e.g., indium tin oxide). In some embodiments, first substrate2404 can have a functionalization chemistry on its surface. In theexamples shown in FIGS. 24A and 24B, first substrate 2404 is overlaidwith a first coating 2406, and first coating 2406 (e.g., a conductivecoating) is further overlaid with a second coating 2408. In someembodiments, first coating 2406 is an indium tin oxide (ITO) coating. Insome embodiments, second coating 2408 is a lawn of capture probes (e.g.,any of the capture probes described herein). In some embodiments, asubstrate can include an ITO coating. In some embodiments, a substratecan include capture probes or capture probes attached to features on thesubstrate.

Biological sample 2402 and second coating 2408 (e.g., a lawn of captureprobes) can be in contact with a permeabilization solution 2410.Non-limiting examples of permeabilization solutions include, enzymes(e.g., proteinase K, pepsin, and collagenase), detergents (e.g., sodiumdodecyl sulfate (SDS), polyethylene glycol tert-octylphenyl ether,polysorbate 80, and polysorbate 20), ribonuclease inhibitors, buffersoptimized for electrophoresis, buffers optimized for permeabilization,buffers optimized for hybridization, or combinations thereof.Permeabilization reagents can also include but are not limited to adried permeabilization reagent, a permeabilization buffer, a bufferwithout a permeabilization reagent, a permeabilization gel, and apermeabilization solution. In some examples, biological samples (e.g.,tissue samples) can be permeabilized first and then be subjected toelectrophoresis.

FIG. 24A shows an example analytical workflow including a first step2412 in which biological sample 2402 can be permeabilized prior tosubjecting the sample 2402 to electrophoresis. Any of thepermeabilization methods disclosed herein can be used during first step2412. Biological sample 2402 includes an analyte 2414. In someembodiments, the analyte 2414 is a negatively charged analyte. Firstsubstrate 2404 can include a capture probe 2416 that is fixed orattached to the first substrate 2404 or attached to features (e.g.,beads) 2418 on the substrate. In some embodiments, capture probe 2416can include any of the capture probes disclosed herein. In someembodiments, first substrate 2402 does not include features and instead,capture probes 2416 are directly attached to the substrate surface. Insome embodiments, the capture probe 2416 is positively charged.

In step 2420, after permeabilization of biological sample 2402concludes, the sample 2402 can be subjected to electrophoresis. Duringelectrophoresis, the biological sample 2402 is subjected to an electricfield that can be generated by sandwiching biological sample 2402between the first substrate 2404 and a second substrate 2422, connectingeach substrate to a cathode and an anode, respectively, and running anelectric current through the substrates. The application of the electricfield “-E” causes the analyte 2404 (e.g., a negatively charged analyte)to migrate towards the substrate 2404 and capture probe 2416 (e.g., apositively charged capture probe) in the direction of the arrows shownin FIG. 24A. In some embodiments, the analyte 2414 migrates towards thecapture probe 2416 for a distance “h.” In some embodiments, the analyte2414 migrates towards a capture probe 2416 through one or morepermeabilized cells within the permeabilized biological sample (e.g.,from an original location in a permeabilized cell to a final location inor close to the capture probe 2416). Second substrate 2422 can includethe first coating 2406 (e.g., a conductive coating), thereby allowingelectric field “-E” to be generated.

In some embodiments, the analyte 2414 is a protein or a nucleic acid. Insome embodiments, the analyte 2414 is a negatively charged protein or anucleic acid. In some embodiments, the analyte 2414 is a positivelycharged protein or a nucleic acid. In some embodiments, the captureprobe 2416 is a protein or a nucleic acid. In some embodiments, thecapture probe 2416 is a positively charged protein or a nucleic acid. Insome embodiments, the capture probe 2416 is a negatively charged proteinor a nucleic acid. In some embodiments, the analyte 2414 is a negativelycharged transcript. In some embodiments, the analyte 2414 is a poly(A)transcript. In some embodiments, the capture probe 2416 is attached to afeature in a feature array. In some embodiments, permeabilizationreagent 2410 can be in contact with sample 2402, first substrate 2404second substrate 2422, or any combination thereof.

FIG. 24B shows an example analytical workflow in which biological sample2402 can be permeabilized and subjected to electrophoresissimultaneously. In some embodiments, simultaneous permeabilization andelectrophoresis of biological sample 2402 can decrease the totalduration of the analytical workflow translating into a more efficientworkflow.

In some embodiments, the permeabilization reaction is conducted at achilled temperature (e.g., about 4° C.). In some embodiments, conductingthe permeabilization reaction at a chilled temperature controls theenzyme activity of the permeabilization reaction. In some embodiments,the permeabilization reaction is conducted at a chilled temperature inorder to prevent drift and/or diffusion of the analyte 2414 from anoriginal location (e.g., a location in a cell of the biological sample2402) until a user is ready to initiate the permeabilization reaction.In some embodiments, the permeabilization reaction is conducted at awarm temperature (e.g., a temperature ranging from about 15° C. to about37° C. or more) in order to initiate and/or increase the rate of thepermeabilization reaction. In some embodiments, once electrophoresis isapplied and/or the permeabilization reaction is heated, thepermeabilization reaction allows for analyte migration from an originallocation (e.g., a location in a cell of the biological sample 2402) tothe capture probe 2416 anchored to the first substrate 2404.

Referring generally to FIGS. 25A-C, example substrate configurations foruse in the active migration of analytes from a first location to asecond location via electrophoresis are shown. FIG. 25A shows an examplesubstrate configuration for use in electrophoresis in which the firstsubstrate 2502 and the second substrate 2522 are aligned at about 90degrees with respect to each other. In this example, the first substrate2502 including biological sample 2504 is placed beneath second substrate2522. Both the first substrate 2502 and the second substrate 2522 can beconnected to electrical wires 2524 that direct an electric current froma power supply to the substrates, thereby generating an electric fieldbetween the substrates. FIG. 25B shows an additional example substrateconfiguration for use during electrophoresis in which the firstsubstrate 2502 and the second substrate 2522 are parallel with respectto each other. In this example, the first substrate 2502 includingbiological sample 2504 is also placed beneath second substrate 2522.

FIG. 25C shows yet an additional example substrate configuration for usein electrophoresis in which the second substrate 2522 and a thirdsubstrate 2526 are aligned at about 90 degrees with respect to the firstsubstrate 2502. Thus, in this example, a first biological sample 2502 aand a second biological sample 2502 b can be subjected toelectrophoresis simultaneously. In some embodiments, 3, 4, 5, 6, 7, 8,9, 10, or more biological samples can be placed on a same substrate andbe subjected to electrophoresis simultaneously. In some embodiments, 2,3, 4, 5, 6, 7, 8, 9, 10, or more top substrates can be placed above asame bottom substrate containing one or more samples in order tosimultaneously subject the one or more samples to electrophoresis. Insome embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more top substrates canbe perpendicularly placed (e.g., at about 90 degrees) above a samebottom substrate containing one or more samples in order tosimultaneously subject the one or more samples to electrophoresis. Insome embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more top substrates canbe placed in a parallel orientation above a same bottom substratecontaining one or more biological samples in order to simultaneouslysubject the one or more samples to electrophoresis. In some embodiments,a configuration of top substrates can be arranged above a same bottomsubstrate containing one or more biological samples in order tosimultaneously subject the one or more samples to electrophoresis. Insome embodiments, a first configuration of top substrates can bearranged above a second array of bottom substrates containing one ormore biological samples in order to simultaneously subject the one ormore biological samples to electrophoresis. In some embodiments,simultaneously subjecting two or more biological samples on a samesubstrate to electrophoresis can provide the advantage of a moreeffective workflow. In some embodiments, one or more of the topsubstrates can contain the biological sample.

In some embodiments, methods utilizing an active capture method caninclude one or more solutions between the biological sample and thesubstrate (e.g., a substrate including capture probes). In someembodiments, the one or more solutions between the biological sample andthe substrate including capture probes can include a permeabilizationbuffer (e.g., any of the permeabilization buffers described herein). Insome embodiments, the one or more solutions between the biologicalsample and the substrate including capture probes can include anelectrophoresis buffer.

In some embodiments, actively capturing analytes can include one or moreporous materials between the biological sample and the substrateincluding capture probes. In some embodiments, the one or more porousmaterials between the biological sample and substrate including captureprobes can include a paper or a blotting membrane. In some embodiments,the one or more porous materials between the biological sample and thesubstrate including capture probes can include a gel containing one ormore solutions. For example, in a non-limiting way, the gel can be aSDS-PAGE gel. In some embodiments, the one or more porous materialsbetween the biological sample and the substrate including capture probescan contain a permeabilization buffer. In some embodiments, the one ormore porous materials between the biological sample and the substrateincluding capture probes can contain an electrophoresis buffer. In someembodiments, actively capturing analytes can include one or moresolutions and one or more porous materials between the biological sampleand the substrate including capture probes.

In some embodiments, the one or more porous materials between thebiological sample and the substrate including capture probes (e.g., anarray) can act as a filter to separate analytes (e.g., analytes ofinterest) from other molecules or analytes present in the biologicalsample. In some embodiments, the analytes (e.g., analytes of interest)are RNA transcripts. In some embodiments, the one or more porousmaterials between the biological sample and the substrate includingcapture probes can act as a filter to separate RNA transcripts fromother molecules (e.g., analytes) such as proteins, lipids and/or othernucleic acids. In some embodiments, the one or more porous materialsbetween the biological sample and the substrate including capture probescan act as a filter to separate the analytes and other molecules basedon physicochemical properties. For example, in a non-limiting way,analytes can be separated on properties such as charge, size (e.g.,length, radius of gyration, effective diameters, etc.), hydrophobicity,hydrophilicity, molecular binding (e.g., immunoaffinity), andcombinations thereof. In some embodiments, the one or more porousmaterials between the biological sample and the substrate includingcapture probes can separate the analytes from other molecules to reducenon-specific binding near the capture probes and therefore improvebinding between the analytes and the capture probes, thus improvingsubsequent assay performance.

In some embodiments, the one or more porous materials between thebiological sample and the substrate including capture probes can act asmolecular sieving matrices for electrophoretic analyte separation. Forexample, in a non-limiting way, separation of analytes can occur basedon physicochemical properties such as charge, size (e.g., length, radiusof gyration, and effective diameters, etc.), electrophoretic mobility,zeta potential, isoelectric point, hydrophobicity, hydrophilicity,molecular binding (e.g., immunoaffinity), and combinations thereof. Insome embodiments, the one or more porous materials between thebiological sample and the substrate including capture probes can be of auniform pore size. In some embodiments, the one or more porous materialsbetween the biological sample and the substrate including capture probescan have discontinuities in pore sizes, as generally used in differentgel electrophoresis schemes. In some embodiments, the one or more porousmaterials between the biological sample and the substrate includingcapture probes can have gradients in pore sizes. For example, the one ormore porous materials (e.g., a hydrogel) can have a gradient of poresizes such that the gradient separates the analytes as the analytesmigrate to the substrate including capture probes (e.g., an array).

In some embodiments, the one or more porous materials between thebiological sample and the substrate including capture probes canseparate the analytes based on length. For example, shorter analyteswill have a higher electrophoretic mobility, and therefore migratefaster towards the capture probes relative to longer analytes in anelectrophoretic setup. In some embodiments, the one or more porousmaterials between the biological sample and the substrate includingcapture probes separate the analytes based on length, such that onlyshorter analytes can migrate through the one or more porous materials toreach the capture probes, while longer analytes cannot reach the captureprobes.

In some embodiments, specific subsets of analytes (e.g., a subset oftranscripts) can be captured by applying an electrophoretic field for acertain amount of time. In some embodiments, specific subsets ofanalytes (e.g., a subset of transcripts) can be captured by selectingdifferent porous materials (e.g., porous materials with differentcompositions) between the biological sample and the substrate includingcapture probes. In some embodiments, specific subsets of analytes (e.g.,a subset of transcripts) can be captured by applying an electrophoreticfield for a certain amount of time and selecting different porousmaterials between the biological sample and the substrate includingcapture probes (e.g., an array).

In some embodiments, the one or more porous materials between thebiological sample and the substrate including capture probes can havediscontinuities in pore sizes that can cause an increase in theconcentration of the migrating analytes (e.g., “stacking”). For example,the one or more porous materials (e.g., a hydrogel) between thebiological sample and the substrate including capture probes can havediscontinuities in pore sizes that can cause an increase in theconcentration of the analytes near the capture probes resulting infavorable binding kinetics and increased sensitivity. In someembodiments, the one or more porous materials between the biologicalsample and the substrate including capture probes can havediscontinuities in pore sizes that enhance the separation betweenmigrating analytes of different sizes and/or lengths. In someembodiments, the one or more porous materials between the biologicalsample and the substrate including capture probes can include a firstporous material and a second porous material, with the first porousmaterial having a larger pore size than the second porous material. Insome embodiments, the first porous material is located on the surface,or near the surface, of the biological sample. In some embodiments, thesecond porous material (e.g., second porous material with a smaller poresize than the first porous material) can be placed on the surface, ornear the surface, of the first porous material. In some embodiments, asanalytes migrate (e.g., migrate via electrophoresis) from the biologicalsample through the first porous material and the second porous materialsequentially, the migrating analytes can collect (e.g., “stack”) at theinterface between the first porous material and the second porousmaterial.

In some embodiments, the one or more porous materials between thebiological sample and the substrate including capture probes can includea gradient in pore sizes for continuous stacking as analytes migratethrough decreasing pore sizes (e.g., decreasing pore diameter). In someembodiments, the one or more porous materials between the biologicalsample and the substrate including capture probes can include a gradientin pore sizes such that the pores decrease in diameter as the analytesmigrate from the biological sample to the substrate including captureprobes. In some embodiments, the pore size gradient can increase theresolution among analytes of different sizes. In some embodiments, thepore size gradient can increase the concentration of the analytes nearthe capture probes. In some embodiments, the pore size gradient cancontinuously reduce the speed at which the analytes migrate and collect(e.g., “stack”) as the analytes migrate through the gradient ofdecreasing pore sizes (e.g., decreasing pore diameter).

In some embodiments, the one or more porous materials between thebiological sample and the substrate including capture probes can includea gradient gel for continuous stacking as analytes migrate throughdecreasing pore sizes (e.g., decreasing pore diameter) of the gradientgel. In some embodiments, the gradient gel can have pores with adecreasing diameter as the analytes migrate toward the capture probes.In some embodiments, the gradient gel can increase the separationresolution among analytes of different sizes. In some embodiments, thegradient gel can increase the concentration of analytes near the captureprobes. In some embodiments, the gradient gel can continuously reducethe speed at which analytes migrate and collect (e.g., “stack”) as theanalytes migrate through the gradient gel of decreasing pore sizes(e.g., decreasing in diameter).

In some embodiments, a biological sample can be placed in a firstsubstrate holder (e.g., a substrate holder described herein). In someembodiments, a spatially-barcoded capture probe array (e.g., captureprobes, barcoded array) can be placed on a second substrate holder(e.g., a substrate holder described herein). In some embodiments, abiological sample can be placed in a first substrate holder that alsocontains capture probes. In some embodiments, the first substrateholder, the second substrate holder, or both can be conductive (e.g. anyof the conductive substrates described herein). In some embodiments, thefirst substrate holder including the biological sample, the secondsubstrate holder including capture probes, or both, can be contactedwith permeabilization reagents (e.g., a permeabilization buffer) andanalytes can be migrated from the biological sample toward the barcodedarray using an electric field.

In some embodiments, electrophoresis can be applied to a biologicalsample on a barcoded array while in contact with a permeabilizationbuffer. In some embodiments, electrophoresis can be applied to abiological sample on a barcoded array while in contact with anelectrophoresis buffer (e.g. a buffer that lacks permeabilizationreagents). In some embodiments, the permeabilization buffer can bereplaced with an electrophoresis buffer after a desired amount of time.In some embodiments, electrophoresis can be applied simultaneously withthe permeabilization buffer or electrophoresis buffer. In someembodiments, electrophoresis can be applied after a desired amount oftime of contact between the biological sample and the permeabilizationbuffer or electrophoresis buffer.

In some embodiments, the biological sample can be placed on a substrate(e.g., a porous membrane, a hydrogel, paper, etc.). In some embodiments,the biological sample placed on the substrate can have a gap (e.g., aspace) between the substrate and the substrate holder (e.g., conductivesubstrate holder). In some embodiments, the barcoded array can be placedon a substrate (e.g., a porous membrane, a hydrogel, paper, etc.). Insome embodiments, the barcoded array can have a gap between thesubstrate and substrate holder (e.g., conductive substrate holder). Insome embodiments, the barcoded array can be placed in direct proximityto the biological sample or at a desired distance from the biologicalsample. In some embodiments, a buffer reservoir can be used between thesubstrate holder (e.g., conductive substrate holder) and the barcodedarray, the biological sample, or both. This setup allows the analytes tobe migrated to a barcoded array while not in proximity with theelectrodes (e.g. conductive substrate holder), thus resulting in morestable electrophoresis.

In some embodiments, a combination of at least two buffers withdifferent ionic compositions can be used to differentially migrateanalytes based on their ionic mobility (e.g., isotachophoresis (ITP)).For example, using two or more buffers with different ionic compositionscan increase the concentration of analytes prior to contact with abarcoded array. Isotachophoresis includes at least two buffers thatcontain a common counter-ion (e.g., ions that have different charge signthan the analytes) and different co-ions (e.g., ions that have the samecharge sign as the analytes) (Smejkal P., et al., Microfluidicisotachophoresis: A review, Electrophoresis, 34.11 1493-1509, (2013)which is incorporated herein by reference in its entirety). In someembodiments, one buffer can contain a co-ion with a higher ionicmobility (e.g. speed at which they travel through solution in anelectric field) than the analytes (e.g., the “leading” buffer). In someembodiments, a second buffer can contain a co-ion with a lower ionicmobility than the analytes (e.g., the “trailing” buffer). In someembodiments, a third buffer can contain a co-ion with an ionic mobilitythat is between the electric mobility of the analytes. In someembodiments, a biological sample can be placed on a first substrateholder (e.g., a conductive substrate holder) and the barcoded array canbe placed on a second substrate holder (e.g., a second conductivesubstrate holder) and contacted with a permeabilization buffer and theanalytes can be migrated away from the biological sample and toward thebarcoded array using an electric field. As the electric field is appliedto the biological sample the analytes can be concentrated in the bufferas they are migrated toward the capture probes. In some embodiments,isotachophoresis can be used with gel-based separations (e.g., any ofthe gel-based separations described herein).

In some embodiments, a permeabilization buffer can be applied to aregion of interest (e.g., region of interest as described herein) in abiological sample. In some embodiments, permeabilization reagents (e.g.a hydrogel containing permeabilization reagents) can be applied to aregion of interest in a biological sample. For example, a region ofinterest can be a region that is smaller in area relative to the overallarea of the biological sample. In some embodiments, the permeabilizationbuffer or permeabilization reagents can be contacted with the biologicalsample and a substrate including capture probes (e.g., an array). Insome embodiments, the biological sample can have more than one region ofinterest (e.g. two, three). In some embodiments, the biological sample,the substrate including capture probes, or both, can be placed in aconductive substrate holder. In some embodiments, analytes can bereleased from the region(s) of interest and migrated from the biologicalsample toward the capture probes with an electric field.

In some embodiments, electrophoretic transfer of analytes can beperformed while retaining the relative spatial locations of analytes ina biological sample while minimizing passive diffusion of an analyteaway from its location in a biological sample. In some embodiments, ananalyte captured by a capture probe (e.g., capture probes on asubstrate) retains the spatial location of the analyte present in thebiological sample from which it was obtained (e.g., the spatial locationof the analyte that is captured by a capture probe on a substrate whenthe analyte is actively migrated to the capture probe by electrophoretictransfer can be more precise or representative of the spatial locationof the analyte in the biological sample than when the analyte is notactively migrated to the capture probe). In some embodiments,electrophoretic transport and binding process is described by theDamköhler number (Da), which is a ratio of reaction and mass transportrates. The fraction of analytes bound and the shape of the biologicalsample will depend on the parameters in the Da. There parameters includeelectromigration velocity U_(e) (depending on analyte electrophoreticmobility μ_(e) and electric field strength E), density of capture probes(e.g., barcoded oligonucleotides) p₀, the binding rate between probes(e.g., barcoded oligonucleotides) and analytes k_(on), and capture areathickness L.

${Da} \sim \frac{k_{on}p_{0}L}{\mu_{e}E}$

Fast migration (e.g., electromigration) can reduce assay time and canminimize molecular diffusion of analytes.

In some embodiments, electrophoretic transfer of analytes can beperformed while retaining the relative spatial alignment of the analytesin the sample. As such, an analyte captured by the capture probes (e.g.,capture probes on a substrate) retains the spatial information of thecell or the biological sample from which it was obtained. Applying anelectrophoretic field to analytes can also result in an increase intemperature (e.g., heat). In some embodiments, the increased temperature(e.g., heat) can facilitate the migration of the analytes towards acapture probe.

In some examples, a spatially-addressable microelectrode array is usedfor spatially-constrained capture of at least one charged analyte ofinterest by a capture probe. For example, a spatially-addressablemicroelectrode array can allow for discrete (e.g., localized)application of an electric field rather than a uniform electric field.The spatially-addressable microelectrode array can be independentlyaddressable. In some embodiments, the electric field can be applied toone or more regions of interest in a biological sample. The electrodesmay be adjacent to each other or distant from each other. Themicroelectrode array can be configured to include a high density ofdiscrete sites having a small area for applying an electric field topromote the migration of charged analyte(s) of interest. For example,electrophoretic capture can be performed on a region of interest using aspatially-addressable microelectrode array.

Schematics illustrating an electrophoretic transfer system configured todirect nucleic acid analytes (e.g., mRNA transcripts) toward aspatially-barcoded capture probe array are shown in FIG. 14A and FIG.14B. In this exemplary configuration of an electrophoretic system, asample 1402 is sandwiched between the cathode 1401 and thespatially-barcoded capture probe array 1404, 1405, and thespatially-barcoded capture probe array 1404, 1405 is sandwiched betweenthe sample 1402 and the anode 1403, such that the sample 1402, 1406 isin contact with the spatially-barcoded capture probes 1407. When anelectric field is applied to the electrophoretic transfer system,negatively charged nucleic acid analytes 1406 will be pulled toward thepositively charged anode 1403 and into the spatially-barcoded array1404, 1405 containing the spatially-barcoded capture probes 1407. Thespatially-barcoded capture probes 1407 interact with the nucleic acidanalytes (e.g., mRNA transcripts hybridize to spatially-barcoded nucleicacid capture probes forming DNA/RNA hybrids) 1406, making the analytecapture more efficient. The electrophoretic system set-up may changedepending on the target analyte. For example, proteins may be positive,negative, neutral, or polar depending on the protein as well as otherfactors (e.g., isoelectric point, solubility, etc.). The skilledpractitioner has the knowledge and experience to arrange theelectrophoretic transfer system to facilitate capture of a particulartarget analyte.

FIG. 15 is an illustration showing an exemplary workflow protocolutilizing an electrophoretic transfer system. In the example, Panel Adepicts a flexible spatially-barcoded feature array being contacted witha sample. The sample can be a flexible array, wherein the array isimmobilized on a hydrogel, membrane, or other flexible substrate. PanelB depicts contact of the array with the sample and imaging of thearray-sample assembly. The image of the sample/array assembly can beused to verify sample placement, choose a region of interest, or anyother reason for imaging a sample on an array as described herein. PanelC depicts application of an electric field using an electrophoretictransfer system to aid in efficient capture of a target analyte. Here,negatively charged mRNA target analytes migrate toward the positivelycharged anode. Panel D depicts application of reverse transcriptionreagents and first strand cDNA synthesis of the captured targetanalytes. Panel E depicts array removal and preparation for libraryconstruction (Panel F) and next-generation sequencing (Panel G).

(vi) Targeted Analysis

In some aspects, arrays (e.g., glass slides) include a plurality ofcapture probes that bind to one or more specific biological targets in asample. The capture probes can be directly or indirectly attached to asubstrate. The capture probe can be or include, for example, DNA or RNA.In some aspects, the capture probes on an array can be immobilized,e.g., attached or bound, to the array via their 5′ or 3′ ends, dependingon the chemical matrix of the array. In some aspects, the probes areattached via a 3′ linkage, thereby leaving a free 5′ end. In someaspects, the probes are attached via a 5′ linkage, thereby leaving afree 3′ end. In some aspects, the probes are immobilized indirectly. Forexample, a probe can be attached to a bead, which bead can be depositedon a substrate. A capture probe as disclosed in this section can includeany of the various components of a capture probe as provided throughoutthis disclosure (e.g., spatial barcodes, UMIs, functional domains,cleavage domains, etc.).

In some aspects, a capture probe or plurality of capture probes interactwith an analyte specific for a particular species or organism (e.g.,host or pathogen). In some aspects, the probe or plurality of probes canbe used to detect a viral, bacterial, or plant protein or nucleic acid.In some aspects, the capture probe or plurality of capture probes can beused to detect the presence of a pathogen (e.g., bacteria or virus) inthe biological sample. In some aspects, the capture probe or pluralityof capture probes can be used to detect the expression of a particularnucleic acid associated with a pathogen (e.g., presence of 16S ribosomalRNA or Human Immunodeficiency Virus (HIV) RNA in a human sample).

In some aspects, the capture domain in the capture probe can interactwith one or more specific analytes (e.g., an analyte or a subset ofanalytes out of the pool of total analytes). The specific analyte(s) tobe detected can be any of a variety of biological molecules includingbut not limited to proteins, nucleic acids, lipids, carbohydrates, ions,small molecules, subcellular targets, or multicomponent complexescontaining any of the above. In some embodiments, the analyte(s) can belocalized to subcellular location(s), including, for example,organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum,chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles,lysosomes, etc. In some embodiments, analyte(s) can be peptides orproteins, including without limitation antibodies and enzymes.

In some aspects, analytes from a biological sample interact with one ormore capture probes (e.g., one or more capture probes immobilizeddirectly or indirectly on a substrate), and the capture probes interactwith specific analytes in the biological sample. In some aspects, thecapture probes are allowed to interact with (e.g., hybridize to)specific analytes, e.g., under appropriate conditions whereoligonucleotide capture probes can hybridize to the target nucleicacids. In some aspects, analytes that did not hybridize to captureprobes are removed (e.g., analytes that do not interact with capturedomains of the capture probes). In some embodiments, removal of analytesthat did not interact with a capture probe can be accomplished by, e.g.,washing the sample to remove such analytes.

In some aspects, a capture probe or plurality of capture probes includesa capture domain that interacts with an analyte or analytes present in abiological sample. In some aspects, the capture probe or plurality ofcapture probes includes a capture domain that detects the presence orlevel amount (e.g., expression level) of a particular analyte oranalytes of interest. The capture domain of a capture probe (immobilizeddirectly or indirectly on a substrate) can be capable of bindingselectively to a desired subtype or subset of nucleic acid. In someaspects, for example, the capture domain binds to a subset of nucleicacids in a genome or a subset of nucleic acids in a transcriptome. Insome aspects, the analyte(s) can include one or more nucleic acids. Insome aspects, the capture probe or plurality of capture probes can beused to detect the expression of a particular transcript (e.g., aparticular mRNA). In some aspects, a capture probe or plurality ofcapture probes can be specific for (e.g., binds to) an individual changein a nucleic acid or protein (e.g., a mutation or single nucleotidepolymorphism (SNP)).

In some aspects, the biological sample includes an analyte that is orincludes a nucleic acid. The nucleic acid can be RNA or DNA. In someaspects, the capture probe or plurality of capture probes detects DNAcopy number of a particular set of nucleic acid analyte or analytes. Forexample, capture probe or plurality of capture probes provided hereincan be used to detect DNA copy number of nucleic acids that sharehomology to each other.

In some aspects, the capture probe or plurality of capture probesincludes a capture domain that detects the presence or level amount(e.g., expression level) of one or more RNA transcripts (e.g., specificRNA transcripts). In some aspects, the capture probe or plurality ofcapture probes includes a capture domain that detects the presence oramount (e.g., expression level) of one or more non-coding RNAs (e.g.,microRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small interferingRNA (siRNA) and small nucleolar RNA (snoRNA). In some aspects, the probeor plurality of probes includes a capture domain that detects thepresence or level amount (e.g., expression level) of one or moreproteins (e.g., proteins expressed of a nucleic acid of interest).

In some aspects, the capture probe or plurality of capture probes can bespecific for a particular protein. In some aspects, the capture probe orplurality of capture probes can be used to detect the presence of aparticular protein of interest. In some aspects, the capture probe orplurality of capture probes can be used to detect translation of aparticular protein. In some aspects, the capture probe or plurality ofcapture probes can specifically interact with an active region of anenzyme, a binding domain of an immunoglobulin, defined domains ofproteins, whole proteins, synthetic peptides, peptides with introducedmutations, aptamer, or any combination thereof. In some aspects, theanalyte(s) can include one or more proteins. In some aspects, theanalyte(s) can include one or more nucleic acids and one or moreproteins.

In some aspects, the capture probe or plurality of capture probes can beused to detect particular post-translational modifications of aparticular protein. In such embodiments, analyte capture agents can bespecific for cell surface analytes having a given state ofposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation),such that a cell surface analyte profile can include posttranslationalmodification information of one or more analytes.

In some aspects, the capture probe or plurality of capture probes can bespecific for a particular set of nucleic acids (e.g., nucleic acids thatare associated with a specific cellular pathway or pathways). In someaspects, the set of nucleic acids is DNA. In some aspects, the set ofnucleic acids is RNA. In some aspects, the set of nucleic acids hassimilar and/or homologous sequences. In some aspects, the set of nucleicacids encodes for analytes that function in a similar cellular pathway.In some aspects, the set of nucleic acids encodes for analytes that areexpressed in a certain pathological state (e.g., cancer, Alzheimer's, orParkinson's disease). In some aspects, the set of nucleic acids encodesfor analytes that are over-expressed in a certain pathological state. Insome aspects, the set of nucleic acids encodes for analytes that areunder-expressed in a certain pathological state.

In some aspects, the capture probe or plurality of capture probes can bespecific for a particular nucleic acid, or detection or expression of aparticular set of proteins (e.g., in a similar cellular pathway). Insome aspects, the set of proteins has similar functional domains. Insome aspects, the set of proteins functions in a similar cellularpathway. In some aspects, the set of proteins is expressed in a certainpathological state (e.g., cancer, Alzheimer's or Parkinson's disease).In some aspects, the set of proteins is over-expressed in a certainpathological state. In some aspects, the set of proteins isunder-expressed in a certain pathological state.

In some embodiments, a capture probe includes a capture domain that iscapable of binding to more than one analyte. In some embodiments, acapture domain can bind to one or more analytes that are about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, about 99% identical, 100% identical to the target analyte. Insome aspects, the capture probe can bind to an analyte that is about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, or about 99% identical to each other. In some embodiments, acapture domain can bind to a conserved region of one or more analytes,in which the conserved regions are about 80% identical, about 85%identical, about 90% identical, about 95% identical, about 96%identical, about 97% identical, about 98% identical, about 99%identical, 100% identical to the target analyte.

In some aspects, a capture probe or plurality of capture probesinteracts with two or more analytes (e.g., nucleic acids or proteins)that are not similar in sequence and/or do not share a conserved domain.In some embodiments, a capture probe includes two or more capturedomains, each of which interacts with a different analyte. In suchembodiments, members of the two or more capture domains can be adjacentto each other in the capture probe and/or members of the two or morecapture domains can be separated from each other in the capture probe byone or more domains (e.g., nucleic acid domains). For example, in someaspects, the sets of analytes that are detected include mutationalchanges in the targeted nucleic acids or proteins. In some aspects, thecapture probe or plurality of capture probes detects sets of nucleicacids or proteins (e.g., non-homologous nucleic acids or proteins) thatare individually mutated during a pathogenic state. In some aspects, thepathogenic state is cancer.

In some aspects, a capture probe or plurality of capture probes includecapture domains that can be used to detect analytes that are typicallydetected using diagnostic panels. In some aspects, the capture probe orplurality of capture probes are used to detect changes in one or moreanalytes. In some aspects, the analyte changes include one or more ofincreased analyte expression, decreased analyte expression, mutatednucleic acid sequences, or any combination thereof. In some aspects, thechanges in the analytes are associated with and/or lead to manifestationof a pathogenic state in a subject. In some aspects, the detectedchanges are compared to a reference analyte or analytes.

(vii) Polypeptide Capture

Provided herein are methods and materials for identifying the locationof a polypeptide in a biological sample. In some embodiments, an analyte(e.g., a polypeptide analyte) can be directly captured on a substrate.For example, polypeptide analytes can be captured by amine groups on afunctionalized substrate. In other examples, an analyte (e.g., apolypeptide analyte) can be captured via an analyte binding moietydirectly attached to a substrate. In some embodiments, the substrate maybe populated with analyte minding moieties directly attached to thesubstrate as well as spatially-barcoded capture probes directly attachedto the substrate. In other embodiments, an analyte (e.g., a polypeptideanalyte) can be captured via an analyte binding moiety indirectlyattached to a substrate. In an example, the substrate may be populatedwith capture probes that are bound to an analyte capture agent, whereinthe analyte capture domain of the analyte capture agent binds to thecapture domain of the capture probe and the analyte binding moiety bindsthe polypeptide analyte.

In some embodiments, an analyte (e.g., a polypeptide analyte) can bedirectly captured or immobilized on a substrate. Direct immobilizationmay be achieved by covalently coupling the polypeptide analyte to thesubstrate via amide bonds between the carboxylic acid of the C-terminalamino acid residue and a functionalized substrate surface. For example,a substrate (e.g., a glass coverslip or slide) can be functionalizedthrough amino-silanization with aminopropyltriethoxysilane. Thesubstrate surfaces are further passivated by overnight incubation withpolyethylene glycol (PEG)-NHS solution, and functionalized slides can bestored in a vacuum desiccator until use. The t-butyloxycarbonylprotecting groups can be removed by incubating the substrate with 90%TFA (v/v in water) for 5 hours before use, thus exposing free aminegroups for peptide immobilization. The resulting functionalizedsubstrate is stable to multiple cycles of Edman degradation and washingsteps.

In some embodiments, methods for capturing polypeptides in a biologicalsample include providing a substrate where an analyte binding moiety isdirectly immobilized on the substrate. In some embodiments, directimmobilization is achieved through chemical modification of thesubstrate and/or chemical modification of the analyte binding moiety.For example, a substrate can be prepared with free amines on thesurface. When exposed to an analyte binding moiety with a freecarboxylic acid on the C-terminal residue, the free amines can formamide bonds with the carboxylic acid thereby covalently coupling theanalyte binding moiety to the substrate. Substrates and/or analytebinding moieties can be modified in any manner that facilitates covalentbonding of the analyte binding moiety to the substrate. Non-limitingexamples of chemical modification that can be used to covalently bindthe analyte binding moiety to the substrate include are describedherein.

In some embodiments, methods for capturing analyte polypeptides includeproviding a substrate (e.g., an array) where the analyte binding moietyis indirectly attached to the substrate. For example, an analyte bindingmoiety can be indirectly attached to a substrate via an oligonucleotide(e.g., a capture agent barcode domain or capture agent barcode domainhybridized to a capture probe) or other domain capable of binding toboth the substrate and the analyte binding domain. The capture agentbarcode domain is described elsewhere herein. The capture agent barcodedomain can be modified to include a cleavage domain, which can attach toa substrate using any of the chemistries described herein. In someembodiments, the capture agent barcode domain can include an analytecapture sequence as described herein, wherein the analyte capturesequence can hybridize to the capture domain of a capture probe. In someembodiments, a substrate (e.g., an array) containing capture probes canbe modified to capture polypeptide analytes by hybridizing the analytecapture sequence of the analyte capture agent to the capture domain of acapture probe.

In some embodiments, methods for capturing analyte polypeptides includeproviding a substrate (e.g., an array) and providing an analyte captureagent to the biological sample. For example, after drying and fixingsectioned tissue samples, the tissue samples can be positioned on asubstrate (e.g., a spatial array), rehydrated, blocked, andpermeabilized (e.g., 3×SSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAseinhibitor for 10 min at 4° C.) before being stained with fluorescentprimary antibodies (1:100) and a pool of analyte capture agents (in3×SSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAse inhibitor for 30 min at 4°C.). The biological sample can be washed, coverslipped (in glycerol+1U/μl RNAse inhibitor), imaged for detected analytes (e.g., using aconfocal microscope or other apparatus capable of fluorescentdetection), and washed again. The analyte-bound analyte capture agentscan be released from the biological sample (e.g., the biological samplecan be treated with proteinase, e.g., proteinase K) and migrated to thespatial array. An analyte capture sequence of the analyte-bound analytecapture agent can be captured by a capture probe capture domain, and thecapture agent barcode domain can be extended to produce aspatially-tagged analyte capture agent. The spatially-tagged analytecapture agents can be processed according to spatial workflows describedherein.

In some embodiments, methods for capturing analyte polypeptides includeproviding blocking probes to analyte capture agents before introducingthe analyte capture agents to a biological sample. In some embodiments,the blocking probes can be alternatively or additionally provided in anyof the rehydrating or blocking buffers provided herein. In someembodiments, the analyte capture agent analyte capture sequence can beblocked prior to binding to the capture probe capture domain using ablocking probe sequence complementary to the analyte capture sequence.Blocking the capture agent barcode domain, particularly the free 3′ endof the capture agent barcode domain (e.g., analyte capture sequence),prior to contacting the analyte capture agents with the biologicalsample and/or substrate, can prevent binding of the analyte capturesequence of the capture agent barcode domains, e.g., prevents thebinding of a poly(A) tail to the capture probe capture domain. In someembodiments, blocking the analyte capture agent analyte capture domainreduces non-specific background staining. In some embodiments, theblocking probes are reversible, such that the blocking probes can beremoved from the analyte capture sequence during or after the time thatanalyte capture agents are in contact with the biological sample. Insome embodiments, the blocking probe can be removing with RNAsetreatment (e.g., RNAse H treatment).

In some embodiments, methods for capturing polypeptides in a biologicalsample include active transfer (e.g., electrophoresis). For example, thebiological sample is placed on a conductive substrate and contacted witha spatial array including one or more analyte binding moieties. Anelectric filed can be applied to the conductive substrate to promotemigration of the polypeptides towards the analyte binding moieties, asdescribed herein.

In some embodiments, methods for identifying the spatial location of apolypeptide in a biological sample include determining the sequence of acaptured polypeptide. In some embodiments, the sequence of the capturedpolypeptide is determined through detection of amino acid residueslabeled with a detectable label (e.g., radiolabel of a fluorophore).Non-limiting examples of detectable labels that can be used forlabelling the captured polypeptide include fluorophores and radiolabels.In some embodiments, the polypeptides are labeled at specific amino acidresidues only (e.g., not all amino acid residues are labeled). In someembodiments, the polypeptide is labeled prior to contacting thebiological sample with the substrate. In some embodiments, a capturedpolypeptide is labeled with fluorophores using standard coupling schemes(see Hernandez et al., New J. Chem. 41:462-469 (2017)). For example,polypeptides may be labeled by reaction with Atto647N-NHS,Atto647Niodoacetamide, TMR-NHS, or JF549-NHS, as appropriate, to labellysines (via NHS) or cysteines (via iodoacetamide). In addition, serineor threonine phosphorylation sites may be selectively labeled via betaelimination followed by conjugate addition via thiols to substitutethiol-linked fluorophores in place of phosphates (see Stevens et al.,Rapid Commun. Mass Specrtom., 15: 2157-2162 (2005)). The number offluorophores incorporated into a polypeptide is any number that may bespectrally resolved. In some instances, four or more fluorophores areutilized.

In some embodiments, a captured polypeptide is radiolabeled. In someembodiments, specific amino acids can be labeled with an isotope.Non-limiting examples of isotopes used to label amino acids include ³H,¹⁴C, ¹⁵N, ³²P, and ¹²⁵I. In some embodiments, the isotope isincorporated into the selected amino acid prior to incorporation into apolypeptide. In some embodiments, the radiolabeled amino acid can beincorporated into the polypeptide after polypeptide formation.

In some embodiments, the sequence of the captured polypeptide isdetermined using Edman degradation (and in some embodiments successiverounds of Edman degradation). In such cases, a polypeptide is captured,and the polypeptide sequence can be resolved by imaging the substratefollowing repeated rounds of Edman degradation. For example, thesubstrate is imaged following each Edman reaction in order to capturethe detectable labels that are produced due to the removal of aminoacids that are a byproduct of the reaction. The information obtained bythe Edman degradation can be complied to identify a polypeptide. In someembodiments, the biological sample is visualized or imaged using lightor fluorescence microscopy.

(viii) Enrichment of Captured Analytes after Capture

In some aspects, spatial analysis of targeted analytes includes anenrichment step or steps post-capture to enrich the captured analytesfor the targeted analyte. For example, the capture domain can beselected or designed for the selective capture of more analytes than thepractitioner desires to analyze. In some embodiments, capture probesthat include random sequences (e.g., random hexamers or similarsequences) that form all or part of the capture domain can be used tocapture nucleic acids from a biological sample in an unbiased way. Forexample, capture probes having capture domains that include randomsequences can be used to generically capture DNA, RNA, or both from abiological sample. Alternatively, capture probes can include capturedomains can that capture mRNA generally. As is well known in the art,this can be on the basis of hybridization to the poly-A tail of mRNAs.In some embodiments, the capture domain includes a sequence thatinteracts with (e.g., hybridizes to) the poly-A tail of mRNAs.Non-limiting examples of such sequences include poly-T DNA sequences andpoly-U RNA sequences. In some embodiments, random sequences can be usedin conjunction with poly-T (or poly-T analogue etc.) sequences. Thus,where a capture domain includes a poly-T (or a “poly-T-like”)oligonucleotide, it can also include a random oligonucleotide sequence.

In some aspects, after capture of more analytes than the practitionerdesires to analyze, methods disclosed herein include enrichment ofparticular captured analytes. In some aspects, methods includeenrichment of analytes that include mutations (e.g., SNPs,) of interest,nucleic acid(s) of interest, and/or proteins(s) of interest.

In some embodiments, methods of spatial analysis provided herein includeselectively enriching one or more analytes of interest (e.g., targetanalytes) after analyte capture. For example, one or more analytes ofinterest can be enriched by addition of one or more oligonucleotides tothe pool of captured analytes. In some embodiments, one or more analytesof interest can be enriched by addition of one or more oligonucleotidesto the pool of captured analytes on the array. In some embodiments, oneor more analytes of interest can be enriched by addition of one or moreoligonucleotides to the pool of captured analytes where the pool ofcaptured analytes have been released (e.g., removed) from the array. Insome embodiments, when captured analytes have been released from thearray the one or more nucleotides can be complementary to a portion of aTSO and R1 sequence, or portion thereof. In some embodiments, theadditional oligonucleotide(s) include a sequence used for priming areaction by a polymerase. For example, one or more primer sequences withsequence complementarity to one or more analytes of interest can be usedto amplify the one or more analyte(s) of interest, thereby selectivelyenriching these analytes. In some embodiments, one or more primersequences can be complementary to other domains on the capture probe(e.g., R1 sequence, or portion thereof, as above), and not complementaryto the analyte. In some embodiments, enrichment by amplification (e.g.,PCR) occurs by using a first primer complementary to an analyte oranalytes of interest (or another domain in the capture probe and theTSO), or complement thereof, and a second primer complementary to aregion of the capture probe, or complement thereof. In some embodiments,the region of the capture probe, or complement thereof, is distal to aspatial barcode from the capture domain, such that enrichment byamplification amplifies both the captured analyte or analytes and its ortheir associated spatial barcodes, thus permitting spatial analysis ofthe enriched analyte or analytes.

In some embodiments, two or more capture probes capture two or moredistinct analytes, which analytes are enriched (e.g., simultaneously orsequentially) from the pool of captured analytes. In some embodiments,enrichment by PCR amplification includes multiple rounds ofamplification. For example, enrichment by PCR amplification can includenested PCR reactions using different primers that are specific for theanalyte or analytes of interest. In some embodiments, enrichment byamplification can be performed using an amplification method that is notPCR. A non-limiting example of a non-PCR amplification method is rollingcircle amplification. Other non-PCR amplification methods are known inthe art.

In some embodiments, an oligonucleotide with sequence complementarity toa captured analyte or analytes of interest, or complement thereof, canbe used to enrich the captured analyte or analytes of interest from thepool of captured analytes. In some embodiments, an oligonucleotide withsequence complementarity to a captured analyte or analytes of interest(or another domain the capture probe), or complement thereof, caninclude one or more functional moieties that are useful in theenrichment process. For example, biotinylated oligonucleotides withsequence complementary to one or more analytes interest, or complementsthereof, can bind to the analyte(s) of interest and can be selectedusing biotinylation-strepavidin affinity using any of a variety ofmethods known in the art (e.g., streptavidin beads). In someembodiments, oligonucleotides with sequence complementary to one or moreanalytes interest, or complements thereof, include a magnetic moiety(e.g., a magnetic bead) that can be used in the enrichment process.

Additionally or alternatively, one or more species of analyte (e.g.,mitochondrial DNA or RNA) can be down-selected (e.g., removed) using anyof a variety of methods. In some embodiments, such down-selection ofanalytes that are not of interest can result in improved capture ofother types of analytes that are of interest. For example, probes can beadministered to a sample that selectively hybridize to ribosomal RNA(rRNA), thereby reducing the pool and concentration of rRNA in thesample. In some embodiments, such down-selection can result in improvedcapture of other types of RNA due to the reduction in non-specific RNApresent in the sample. Additionally or alternatively, duplex-specificnuclease (DSN) treatment can remove rRNA (see, e.g., Archer, et al,Selective and flexible depletion of problematic sequences from RNA-seqlibraries at the cDNA stage, BMC Genomics, 15 401, (2014), the entirecontents of which is incorporated herein by reference). In someembodiments, hydroxyapatite chromatography can be used to removeabundant species (e.g., rRNA).

(ix) RNA-Templated Ligation

In some embodiments of methods provided here, RNA-templated ligation isused to interrogate spatial gene expression in a biological sample(e.g., an FFPE tissue section). RNA-templated ligation enables sensitivemeasurement of specific nucleic acid analytes of interest that otherwisemight be analyzed less sensitively with a whole transcriptomic approach.It provides advantages of compatibility with common histochemical stainsand suitability for analysis of decade-old materials (e.g., FFPEsamples) and exceedingly small microdissected tissue fragments.

In some aspects, the steps of RNA-templated ligation include: (1)hybridization of pairs of probes (e.g., DNA probes) to RNA (e.g.,formalin fixed RNA) within a tissue section; (2) ligation of adjacentlyannealed probe pairs in situ; (3) RNase H treatment that (i) releasesRNA-templated ligation products from the tissue (e.g., into solution)for downstream analysis and (ii) destroys unwanted DNA-templatedligation products; and optionally, (4) amplification of RNA-templatedligation products (e.g., by multiplex PCR).

In some aspects, disclosed herein are methods of direct detection of RNAtarget-DNA probe duplexes without first converting RNA to cDNA byreverse transcription. In some aspects, RNA-templated ligation caninclude a DNA ligase. In some aspects, RNA-templated ligation caninclude RNA ligase. In some aspects, RNA-templated ligation can includeT4 RNA ligase.

In some aspects, RNA-templated ligation is used for detection of RNA,determination of RNA sequence identity, and/or expression monitoring andtranscript analysis. In some aspects, RNA-templated ligation allows fordetection of a particular change in a nucleic acid (e.g., a mutation orsingle nucleotide polymorphism (SNP)), detection or expression of aparticular nucleic acid, or detection or expression of a particular setof nucleic acids (e.g., in a similar cellular pathway or expressed in aparticular pathology). In some embodiments, the methods that includeRNA-templated ligation are used to analyze nucleic acids, e.g., bygenotyping, quantitation of DNA copy number or RNA transcripts,localization of particular transcripts within samples, and the like. Insome aspects, systems and methods provided herein that includeRNA-templated ligation identify single nucleotide polymorphisms (SNPs).In some aspects, such systems and methods identify mutations.

In some aspects, disclosed herein are methods of detecting RNAexpression that include bringing into contact a first probe, a secondprobe, and ligase (e.g., T4 RNA ligase). In some embodiments, the firstprobe and the second probe are designed to hybridize to a targetsequence such that the 5′ end of the first probe and the 3′ end of thesecond probe are adjacent and can be ligated, wherein at least the5′-terminal nucleotide of the first probe and at least the 3′-terminalnucleotide of the second probe are deoxyribonucleotides (DNA), andwherein the target sequence includes (e.g., is composed of)ribonucleotides (RNA). After hybridization, a ligase (e.g., T4 RNAligase) ligates the first probe and the second probe if the targetsequence is present in the target sample, but does not ligate the firstprobe and the second probe if the target sequence is not present in thetarget sample. The presence or absence of the target sequence in thebiological sample can be determined by determining whether or not thefirst and second probes were ligated in the presence of ligase. Any of avariety of methods can be used to determine whether or not the first andsecond probes were ligated in the presence of ligase, including but notlimited to, sequencing the ligated product, hybridizing the ligatedproduct with a detection probe that hybridizes only when the first andsecond probes were ligated in the presence of ligase, restriction enzymeanalysis, and other methods known in the art.

In some aspects, two or more RNA analytes are analyzed using methodsthat include RNA-templated ligation. In some aspects, when two or moreanalytes are analyzed, a first and second probe that is specific for(e.g., specifically hybridizes to) each RNA analyte are used.

In some aspects, three or more probes are used in RNA-templated ligationmethods provided herein. In some embodiments, the three or more probesare designed to hybridize to a target sequence such that the three ormore probes hybridize adjacent to each other such that the 5′ and 3′ends of adjacent probes can be ligated. In some embodiments, thepresence or absence of the target sequence in the biological sample canbe determined by determining whether or not the three or more probeswere ligated in the presence of ligase.

In some aspects, the first probe is a DNA probe. In some aspects, thefirst probe is a chimeric DNA/RNA probe. In some aspects, the secondprobe is a DNA probe. In some aspects, the second probe is a chimericDNA/RNA probe.

In some aspects, methods of RNA-templated ligation utilize the T4 RNALigase 2 to efficiently join adjacent chimeric RNA-DNA probe pairshybridized in situ on fixed RNA target sequences. Subsequent treatmentwith RNase H releases RNA-templated ligation products (e.g., intosolution) for downstream analysis.

(x) Region of Interest

A biological sample can have regions that show morphological feature(s)that may indicate the presence of disease or the development of adisease phenotype. For example, morphological features at a specificsite within a tumor biopsy sample can indicate the aggressiveness,therapeutic resistance, metastatic potential, migration, stage,diagnosis, and/or prognosis of cancer in a subject. A change in themorphological features at a specific site within a tumor biopsy sampleoften correlate with a change in the level or expression of an analytein a cell within the specific site, which can, in turn, be used toprovide information regarding the aggressiveness, therapeuticresistance, metastatic potential, migration, stage, diagnosis, and/orprognosis of cancer in a subject. A region or area within a biologicalsample that is selected for specific analysis (e.g., a region in abiological sample that has morphological features of interest) is oftendescribed as “a region of interest.”

A region of interest in a biological sample can be used to analyze aspecific area of interest within a biological sample, and thereby, focusexperimentation and data gathering to a specific region of a biologicalsample (rather than an entire biological sample). This results inincreased time efficiency of the analysis of a biological sample.

A region of interest can be identified in a biological sample using avariety of different techniques, e.g., expansion microscopy, brightfield microscopy, dark field microscopy, phase contrast microscopy,electron microscopy, fluorescence microscopy, reflection microscopy,interference microscopy, confocal microscopy, and visual identification(e.g., by eye), and combinations thereof. For example, the staining andimaging of a biological sample can be performed to identify a region ofinterest. In some examples, the region of interest can correspond to aspecific structure of cytoarchitecture. In some embodiments, abiological sample can be stained prior to visualization to providecontrast between the different regions of the biological sample. Thetype of stain can be chosen depending on the type of biological sampleand the region of the cells to be stained. In some embodiments, morethan one stain can be used to visualize different aspects of thebiological sample, e.g., different regions of the sample, specific cellstructures (e.g., organelles), or different cell types. In otherembodiments, the biological sample can be visualized or imaged withoutstaining the biological sample.

In some embodiments, staining and imaging a biological sample prior tocontacting the biological sample with a spatial array is performed toselect samples for spatial analysis. In some embodiments, the stainingincludes applying a fiducial marker as described herein, includingfluorescent, radioactive, chemiluminescent, or colorimetric detectablemarkers. In some embodiments, the staining and imaging of biologicalsamples allows the user to identify the specific sample (or region ofinterest) the user wishes to assess.

In some examples, an array (e.g., any of the exemplary arrays describedherein) can be contacted with only a portion of a biological sample(e.g., a cell, a tissue section, or a region of interest). In someexamples, a biological sample is contacted with only a portion of anarray (e.g., any of the exemplary arrays described herein). In someembodiments, capture probes on an array corresponding to regions ofinterest of a biological sample (e.g., proximal to the region ofinterest) can be selectively cleaved and analyzed. For example, captureprobes on an array may be deactivated or eliminated outside of areascorresponding to regions of interest of a biological sample. In someembodiments, capture probes including a photocleavable bond and on thearray in areas corresponding to regions of interest of a biologicalsample can be selectively cleaved by using light. A mirror, mirrorarray, a lens, a moving stage, and/or a photomask can be used to directthe light to regions of the array that correspond to areas outside oneor more regions of interest in the biological sample. Some embodimentsinclude deactivating or eliminating capture probes, e.g., capture probescomprising a photocleavable bond as described herein, using light. Insome embodiments, a laser, e.g., a scanning laser, can be used todeactivate or eliminate capture probes. In some embodiments, theeliminated member of the plurality of capture probes can be washed away.In some embodiments, regions of interest can be labeled with differentheavy metals, and a laser can sequentially ablate these regions ofinterest before mass spectrometry identification. A laser can, forexample, deactivate or eliminate capture probes through UV lightdestruction of DNA, heat, inducing a chemical reaction that prevents thecapture probes from moving to the next step, inducing photocleavage of aphotocleavable bond, or a combination thereof. In some examples, aportion of the array can be deactivated such that it does not interactwith the analytes in the biological sample (e.g., optical deactivation,chemical deactivation, heat deactivation, or blocking of the captureprobes in the array (e.g., using blocking probes)). In some embodiments,the capture probes can be blocked (e.g., masked or modified) prior tocontacting the biological sample with the array. For example, the free3′ end of the capture probe can be blocked or modified prior tocontacting the biological sample with the array to avoid modification ofthe capture probes (e.g., to avoid the removal or modification or thefree 3′ OH group on the end of the capture probes). In some embodiments,the capture probes can be blocked prior to contacting the biologicalsample to the array. In some embodiments, the blocking probe is used toblock or modify the free 3′ end of the capture domain of the captureprobe. In some embodiments, the blocking probes can be hybridized to thecapture probe. In some embodiments, the free 3′ end of the capturedomain can be blocked by chemical modification.

In some examples, a region of interest can be removed from a biologicalsample and then the region of interest can be contacted to the array(e.g., any of the arrays described herein). A region of interest can beremoved from a biological sample using microsurgery, laser capturemicrodissection, chunking, a microtome, dicing, trypsinization,labelling, and/or fluorescence-assisted cell sorting, and the like. Insome embodiments, the biological sample is dissected using laser capturemicrodissection, retaining one or more portions of biological sample foranalysis and/or discarding one or more portions of biological sample. Insome embodiments, the biological sample is dissected on the array. Insome embodiments, one or more regions of interest are selected usingspatially addressable microelectrode arrays.

In some examples, a region of interest can be permeabilized or lysedwhile areas outside the region of interest are not permeabilized orlysed (e.g., Kashyap et al. Sci Rep. 2016; 6: 29579, herein incorporatedby reference in its entirety). For example, in some embodiments, aregion of interest can be contacted with a hydrogel comprising apermeabilization or lysing reagent. In some embodiments, the area(s)outside the region of interest are not contacted with the hydrogelcomprising the permeabilization or lysing reagent. In some embodiments,the eliminated members of the plurality of capture probes are washedaway after the permeabilization of the biological sample.

(f) Partitioning

As discussed above, in some embodiments, the sample can optionally beseparated into single cells, cell groups, or other fragments/pieces thatare smaller than the original, unfragmented sample. Each of thesesmaller portions of the sample can be analyzed to obtainspatially-resolved analyte information for the sample.

For samples that have been separated into smaller fragments—andparticularly, for samples that have been disaggregated, dissociated, orotherwise separated into individual cells—one method for analyzing thefragments involves separating the fragments into individual partitions(e.g., fluid droplets), and then analyzing the contents of thepartitions. In general, each partition maintains separation of its owncontents from the contents of other partitions. The partition can be adroplet in an emulsion, for example.

The partitions can be flowable within fluid streams. The partitions caninclude, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionscan include a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions can beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, the entire contents of whichare incorporated herein by reference. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are described,for example, in U.S. Patent Application Publication No. 2010/0105112,the entire contents of which are incorporated herein by reference.

For droplets in an emulsion, allocating individual particles to discretepartitions can be accomplished, for example, by introducing a flowingstream of particles in an aqueous fluid into a flowing stream of anon-aqueous fluid, such that droplets are generated at the junction ofthe two streams. Fluid properties (e.g., fluid flow rates, fluidviscosities, etc.), particle properties (e.g., volume fraction, particlevolume, particle concentration, etc.), microfluidic architectures (e.g.,channel geometry, etc.), and other parameters can be adjusted to controlthe occupancy of the resulting partitions (e.g., number of analytes perpartition, number of beads per partition, etc.). For example, partitionoccupancy can be controlled by providing the aqueous stream at a certainconcentration and/or flow rate of analytes.

To generate single analyte partitions, the relative flow rates of theimmiscible fluids can be selected such that, on average, the partitionscan contain less than one analyte per partition to ensure that thosepartitions that are occupied are primarily singly occupied. In somecases, partitions among a plurality of partitions can contain at mostone analyte. In some embodiments, the various parameters (e.g., fluidproperties, particle properties, microfluidic architectures, etc.) canbe selected or adjusted such that a majority of partitions are occupied,for example, allowing for only a small percentage of unoccupiedpartitions. The flows and channel architectures can be controlled as toensure a given number of singly occupied partitions, less than a certainlevel of unoccupied partitions and/or less than a certain level ofmultiply occupied partitions.

The channel segments described herein can be coupled to any of a varietyof different fluid sources or receiving components, includingreservoirs, tubing, manifolds, or fluidic components of other systems.As will be appreciated, the microfluidic channel structure can have avariety of geometries. For example, a microfluidic channel structure canhave one or more than one channel junction. As another example, amicrofluidic channel structure can have 2, 3, 4, or 5 channel segmentseach carrying particles that meet at a channel junction. Fluid can bedirected to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can include compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid can also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary, and/or gravity flow.

In addition to cells and/or analytes, a partition can include additionalcomponents, and in particular, one or more beads. A partition caninclude a single gel bead, a single cell bead, or both a single cellbead and single gel bead. A variety of different beads can beincorporated into partitions. In some embodiments, for example,non-barcoded beads can be incorporated into the partitions. For example,where the biological particle (e.g., a cell) that is incorporated intothe partitions carries one or more barcodes (e.g., spatial barcode(s),UMI(s), and combinations thereof), the bead can be a non-barcoded bead.

In some embodiments, a barcode carrying bead can be incorporated intopartitions. In general, an individual bead can be coupled to any numberof individual nucleic acid molecules, for example, from one to tens tohundreds of thousands or even millions of individual nucleic acidmolecules. The respective barcodes for the individual nucleic acidmolecules can include both common sequence segments or relatively commonsequence segments and variable or unique sequence segments betweendifferent individual nucleic acid molecules coupled to the same bead.For example, a nucleic acid molecule (e.g., an oligonucleotide), can becoupled to a bead by a releasable linkage (e.g., a disulfide linker),wherein the nucleic acid molecule can be or include a barcode. Forexample, barcodes can be injected into droplets previous to, subsequentto, or concurrently with droplet generation. The delivery of thebarcodes to a particular partition allows for the later attribution ofthe characteristics of the individual biological particle to theparticular partition. Barcodes can be delivered, for example on anucleic acid molecule (e.g., an oligonucleotide), to a partition via anysuitable mechanism. Barcoded nucleic acid molecules can be delivered toa partition via a microcapsule. A microcapsule, in some instances, caninclude a bead. The same bead can be coupled (e.g., via releasablelinkage) to one or more other nucleic acid molecules.

In some embodiments, a microcapillary array with spatially barcodedbeads can be generated. A plurality of spatially barcoded beads can beflowed into channels on a microcapillary array such that eachmicrocapillary channel can be loaded with one spatially barcoded bead.In some embodiments, the spatially barcoded bead microcapillary arraycan be contacted to a biological sample for subsequent spatial analysisof biological analytes within the biological sample. In someembodiments, a microcapillary array channel can mechanically compressthe biological sample and form fluidically isolated reaction chambers.In some embodiments, reagents (e.g., enzymes, nucleic acids) areintroduced into the reaction chambers. The reagents can be sealed (e.g.,by silicone oil, mineral oil) within the reaction chambers andincubated, allowing for a cellular and/or nuclear permeabilizationreaction to occur. In some embodiments, biological analytes (e.g., DNA,RNA, proteins, metabolites, small molecules, and lipids) are releasedand captured onto the spatially barcoded microcapillary array,preserving their spatial information. In some embodiments, spatialanalysis using a spatially barcoded feature microcapillary array can beused to obtain spatial information of the biological sample analytes atsingle-cell resolution.

The nucleic acid molecule can include a functional domain that can beused in subsequent processing. For example, the functional domain caninclude one or more of a sequencer specific flow cell attachmentsequence (e.g., a P5 sequence for Illumina® sequencing systems) and asequencing primer sequence (e.g., a R1 primer for Illumina® sequencingsystems). The nucleic acid molecule can include a barcode sequence foruse in barcoding the sample (e.g., DNA, RNA, protein, etc.). In somecases, the barcode sequence can be bead-specific such that the barcodesequence is common to all nucleic acid molecules coupled to the samebead. Alternatively or in addition, the barcode sequence can bepartition-specific such that the barcode sequence is common to allnucleic acid molecules coupled to one or more beads that are partitionedinto the same partition. The nucleic acid molecule can include aspecific priming sequence, such as an mRNA specific priming sequence(e.g., poly(T) sequence), a targeted priming sequence, and/or a randompriming sequence. The nucleic acid molecule can include an anchoringsequence to ensure that the specific priming sequence hybridizes at thesequence end (e.g., of the mRNA). For example, the anchoring sequencecan include a random short sequence of nucleotides, such as a 1-mer,2-mer, 3-mer or longer sequence, which can ensure that a poly(T) segmentis more likely to hybridize at the sequence end of the poly(A) tail ofthe mRNA.

The nucleic acid molecule can include a unique molecular identifyingsequence (e.g., unique molecular identifier (UMI)). In some embodiments,the unique molecular identifying sequence can include from about 5 toabout 8 nucleotides. Alternatively, the unique molecular identifyingsequence can include less than about 5 or more than about 8 nucleotides.The unique molecular identifying sequence can be a unique sequence thatvaries across individual nucleic acid molecules coupled to a singlebead. In some embodiments, the unique molecular identifying sequence canbe a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI can provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantitation of the numberof original expressed RNA.

A partition can also include one or more reagents. Unique identifiers,such as barcodes, can be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead). Microfluidic channel networks (e.g., on achip) can be utilized to generate partitions. Alternative mechanisms canalso be employed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

In some embodiments, barcoded nucleic acid molecules can be initiallyassociated with a microcapsule and then released from the microcapsule.Release of the barcoded nucleic acid molecules can be passive (e.g., bydiffusion out of the microcapsule). In addition or alternatively,release from the microcapsule can be upon application of a stimuluswhich allows the barcoded nucleic acid nucleic acid molecules todissociate or to be released from the microcapsule. Such stimulus candisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

In some embodiments, one more barcodes (e.g., spatial barcodes, UMIs, ora combination thereof) can be introduced into a partition as part of theanalyte. As described previously, barcodes can be bound to the analytedirectly, or can form part of a capture probe or analyte capture agentthat is hybridized to, conjugated to, or otherwise associated with ananalyte, such that when the analyte is introduced into the partition,the barcode(s) are introduced as well. As described above, FIG. 16 showsan example of a microfluidical channel structure for partitioningindividual analytes (e.g., cells) into discrete partitions.

FIG. 16 shows an example of a microfluidic channel structure forpartitioning individual analytes (e.g., cells) into discrete partitions.The channel structure can include channel segments 1601, 1602, 1603, and1604 communicating at a channel junction 1605. In operation, a firstaqueous fluid 1606 that includes suspended biological particles (orcells) 1607 may be transported along channel segment 1601 into junction1605, while a second fluid 1608 that is immiscible with the aqueousfluid 1606 is delivered to the junction 1605 from each of channelsegments 1602 and 1603 to create discrete droplets 1609, 1610 of thefirst aqueous fluid 1606 flowing into channel segment 1604, and flowingaway from junction 1605. The channel segment 1604 may be fluidicallycoupled to an outlet reservoir where the discrete droplets can be storedand/or harvested. A discrete droplet generated may include an individualbiological particle 1607 (such as droplets 1609). A discrete dropletgenerated may include more than one individual biological particle 1607.A discrete droplet may contain no biological particle 1607 (such asdroplet 1610). Each discrete partition may maintain separation of itsown contents (e.g., individual biological particle 1607) from thecontents of other partitions.

FIG. 17A shows another example of a microfluidic channel structure 1700for delivering beads to droplets. The channel structure includes channelsegments 1701, 1702, 1703, 1704 and 1705 communicating at a channeljunction 1706. During operation, the channel segment 1701 can transportan aqueous fluid 1707 that includes a plurality of beads 1708 along thechannel segment 1701 into junction 1706. The plurality of beads 1708 canbe sourced from a suspension of beads. For example, the channel segment1701 can be connected to a reservoir that includes an aqueous suspensionof beads 1708. The channel segment 1702 can transport the aqueous fluid1707 that includes a plurality of particles 1709 (e.g., cells) along thechannel segment 1702 into junction 1706. In some embodiments, theaqueous fluid 1707 in either the first channel segment 1701 or thesecond channel segment 1702, or in both segments, can include one ormore reagents, as further described below.

A second fluid 1710 that is immiscible with the aqueous fluid 1707(e.g., oil) can be delivered to the junction 1706 from each of channelsegments 1703 and 1704. Upon meeting of the aqueous fluid 1707 from eachof channel segments 1701 and 1702 and the second fluid 1710 from each ofchannel segments 1703 and 1704 at the channel junction 1706, the aqueousfluid 1707 can be partitioned as discrete droplets 1711 in the secondfluid 1710 and flow away from the junction 1706 along channel segment1705. The channel segment 1705 can deliver the discrete droplets to anoutlet reservoir fluidly coupled to the channel segment 1705, where theycan be harvested.

As an alternative, the channel segments 1701 and 1702 can meet atanother junction upstream of the junction 1706. At such junction, beadsand biological particles can form a mixture that is directed alonganother channel to the junction 1706 to yield droplets 1711. The mixturecan provide the beads and biological particles in an alternatingfashion, such that, for example, a droplet includes a single bead and asingle biological particle.

The second fluid 1710 can include an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets1711.

The partitions described herein can include small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL,20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

In the foregoing discussion, droplets with beads were formed at thejunction of different fluid streams. In some embodiments, droplets canbe formed by gravity-based partitioning methods.

FIG. 17B shows a cross-section view of another example of a microfluidicchannel structure 1750 with a geometric feature for controlledpartitioning. A channel structure 1750 can include a channel segment1752 communicating at a channel junction 1758 (or intersection) with areservoir 1754. In some instances, the channel structure 1750 and one ormore of its components can correspond to the channel structure 1700 andone or more of its components.

An aqueous fluid 1760 comprising a plurality of particles 1756 may betransported along the channel segment 1752 into the junction 1758 tomeet a second fluid 1762 (e.g., oil, etc.) that is immiscible with theaqueous fluid 1760 in the reservoir 1754 to create droplets 1764 of theaqueous fluid 1760 flowing into the reservoir 1754. At the junction 1758where the aqueous fluid 1760 and the second fluid 1762 meet, dropletscan form based on factors such as the hydrodynamic forces at thejunction 1758, relative flow rates of the two fluids 1760, 1762, fluidproperties, and certain geometric parameters (e.g., Δh, etc.) of thechannel structure 1750. A plurality of droplets can be collected in thereservoir 1754 by continuously injecting the aqueous fluid 1760 from thechannel segment 1752 at the junction 1758.

A discrete droplet generated may comprise one or more particles of theplurality of particles 1756. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 1760 can have a substantiallyuniform concentration or frequency of particles 1756. As describedelsewhere herein, the particles 1756 (e.g., beads) can be introducedinto the channel segment 1752 from a separate channel (not shown in FIG.17). The frequency of particles 1756 in the channel segment 1752 may becontrolled by controlling the frequency in which the particles 1756 areintroduced into the channel segment 1752 and/or the relative flow ratesof the fluids in the channel segment 1752 and the separate channel. Insome instances, the particles 1756 can be introduced into the channelsegment 1752 from a plurality of different channels, and the frequencycontrolled accordingly. In some instances, different particles may beintroduced via separate channels. For example, a first separate channelcan introduce beads and a second separate channel can introducebiological particles into the channel segment 1752. The first separatechannel introducing the beads may be upstream or downstream of thesecond separate channel introducing the biological particles.

In some instances, the second fluid 1762 may not be subjected to and/ordirected to any flow in or out of the reservoir 1754. For example, thesecond fluid 1762 may be substantially stationary in the reservoir 1754.In some instances, the second fluid 1762 may be subjected to flow withinthe reservoir 1754, but not in or out of the reservoir 1754, such as viaapplication of pressure to the reservoir 1754 and/or as affected by theincoming flow of the aqueous fluid 1760 at the junction 1758.Alternatively, the second fluid 1762 may be subjected and/or directed toflow in or out of the reservoir 1754. For example, the reservoir 1754can be a channel directing the second fluid 1762 from upstream todownstream, transporting the generated droplets.

The channel structure 1750 at or near the junction 1758 may have certaingeometric features that at least partly determine the volumes and/orshapes of the droplets formed by the channel structure 1750. The channelsegment 1752 can have a first cross-section height, h1, and thereservoir 1754 can have a second cross-section height, h2. The firstcross-section height, h1, and the second cross-section height, h2, maybe different, such that at the junction 1758, there is a heightdifference of Δh. The second cross-section height, h2, may be greaterthan the first cross-section height, h1. In some instances, thereservoir may thereafter gradually increase in cross-section height, forexample, the more distant it is from the junction 1758. In someinstances, the cross-section height of the reservoir may increase inaccordance with expansion angle, β, at or near the junction 1758. Theheight difference, Δh, and/or expansion angle, β, can allow the tongue(portion of the aqueous fluid 1760 leaving channel segment 1752 atjunction 1758 and entering the reservoir 1754 before droplet formation)to increase in depth and facilitate decrease in curvature of theintermediately formed droplet. For example, droplet volume may decreasewith increasing height difference and/or increasing expansion angle.

In some instances, the flow rate of the aqueous fluid 1760 entering thejunction 1758 can be between about 0.04 microliters (μL)/minute (min)and about 40 μL/min. In some instances, the flow rate of the aqueousfluid 1760 entering the junction 1758 can be between about 0.01microliters (μL)/minute (min) and about 100 μL/min. Alternatively, theflow rate of the aqueous fluid 1760 entering the junction 1758 can beless than about 0.01 μL/min. alternatively, the flow rate of the aqueousfluid 1760 entering the junction 1758 can be greater than about 40μL/min, such as 45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min,70 μL/min, 75 μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100μL/min, 110 μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, orgreater. At lower flow rates, such as flow rates of about less than orequal to 10 microliters/minute, the droplet radius may not be dependenton the flow rate of the aqueous fluid 1760 entering the junction 1758.The second fluid 1762 may be stationary, or substantially stationary, inthe reservoir 1754. Alternatively, the second fluid 1762 may be flowing,such as at the above flow rates described for the aqueous fluid 1760.

While FIG. 17B illustrates the height difference, Δh, being abrupt atthe junction 1758 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 1758, a bottom wall ofthe channel and a bottom wall of the reservoir can meet at an anglegreater than 90°. Alternatively or in addition, a top wall (e.g.,ceiling) of the channel and a top wall (e.g., ceiling) of the reservoircan meet an angle greater than 90°. A gradual increase or decrease maybe linear or non-linear (e.g., exponential, sinusoidal, etc.).Alternatively or in addition, the height difference may variablyincrease and/or decrease linearly or non-linearly. While FIG. 17Billustrates the expanding reservoir cross-section height as linear(e.g., constant expansion angle, β), the cross-section height may expandnon-linearly. For example, the reservoir may be defined at leastpartially by a dome-like (e.g., hemispherical) shape having variableexpansion angles. The cross-section height may expand in any shape.

FIG. 17C depicts a workflow wherein cells are partitioned into dropletsalong with barcode-bearing beads 1770. See FIG. 17A. The droplet formsan isolated reaction chamber wherein the cells can be lysed 1771 andtarget analytes within the cells can then be captured 1772 and amplified1773, 1774 according to previously described methods. After sequencelibrary preparation clean-up 1775, the material is sequenced and/orquantified 1776 according to methods described herein. For example, theworkflow shown in FIG. 17C can be used with a biological sample on anarray, where the features of the array have been delivered to thesubstrate via a droplet manipulation system. In some embodiments,capture probes on the features can specifically bind analytes present inthe biological sample. In some embodiments, the features can be removedfrom the substrate (e.g., removed by any method described herein) andpartitioned into droplets with barcode-bearing beads for furtheranalysis according to methods described herein.

It should be noted that while the example workflow in FIG. 17C includessteps specifically for the analysis of mRNA, analogous workflows can beimplemented for a wide variety of other analytes, including any of theanalytes described previously.

By way of example, in the context of analyzing sample RNA as shown inFIG. 17C, the poly(T) segment of one of the released nucleic acidmolecules (e.g., from the bead) can hybridize to the poly(A) tail of amRNA molecule. Reverse transcription can result in a cDNA transcript ofthe mRNA, which transcript includes each of the sequence segments of thenucleic acid molecule. If the nucleic acid molecule includes ananchoring sequence, it will more likely hybridize to and prime reversetranscription at the sequence end of the poly(A) tail of the mRNA.

Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules can include a common barcode sequence segment.However, the transcripts made from the different mRNA molecules within agiven partition can vary at the unique molecular identifying sequencesegment (e.g., UMI segment). Beneficially, even following any subsequentamplification of the contents of a given partition, the number ofdifferent UMIs can be indicative of the quantity of mRNA originatingfrom a given partition. As noted above, the transcripts can beamplified, cleaned up and sequenced to identify the sequence of the cDNAtranscript of the mRNA, as well as to sequence the barcode segment andthe UMI segment. While a poly(T) primer sequence is described, othertargeted or random priming sequences can also be used in priming thereverse transcription reaction. Likewise, although described asreleasing the barcoded oligonucleotides into the partition, in somecases, the nucleic acid molecules bound to the bead can be used tohybridize and capture the mRNA on the solid phase of the bead, forexample, in order to facilitate the separation of the RNA from othercell contents.

In some embodiments, partitions include precursors that include afunctional group that is reactive or capable of being activated suchthat it becomes reactive can be polymerized with other precursors togenerate gel beads that include the activated or activatable functionalgroup. The functional group can then be used to attach additionalspecies (e.g., disulfide linkers, primers, other oligonucleotides, etc.)to the gel beads. For example, some precursors featuring a carboxylicacid (COOH) group can co-polymerize with other precursors to form a beadthat also includes a COOH functional group. In some cases, acrylic acid(a species comprising free COOH groups), acrylamide, andbis(acryloyl)cystamine can be co-polymerized together to generate a beadwith free COOH groups. The COOH groups of the bead can be activated(e.g., via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

In some embodiments, a bead can be formed from materials that includedegradable chemical cross-linkers, such as BAC or cystamine. Degradationof such degradable cross-linkers can be accomplished through a number ofmechanisms. In some examples, a bead can be contacted with a chemicaldegrading agent that can induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent can be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent can degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead.

A degradable bead can include one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimulus,the bond is broken and the bead degrades within the partition. Thelabile bond can be a chemical bond (e.g., covalent bond, ionic bond) orcan be another type of physical interaction (e.g., van der Waalsinteractions, dipole-dipole interactions, etc.). In some embodiments, across-linker used to generate a bead can include a labile bond. Uponexposure to the appropriate conditions, the labile bond can be brokenand the bead degraded. For example, a polyacrylamide bead featuringcystamine and linked, via a disulfide bond, to a barcode sequence, canbe combined with a reducing agent within a droplet of a water-in-oilemulsion. Within the droplet, the reducing agent can break the variousdisulfide bonds, resulting in bead degradation and release of thebarcode sequence into the aqueous, inner environment of the droplet. Inanother example, heating of a droplet with a bead-bound barcode sequencein basic solution can also result in bead degradation and release of theattached barcode sequence into the aqueous, inner environment of thedroplet. The free species (e.g., oligonucleotides, nucleic acidmolecules) can interact with other reagents contained in the partition.

A degradable bead can be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc.) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species can have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some embodiments, a species can also be attached to a degradable beadvia a degradable linker (e.g., disulfide linker). The degradable linkercan respond to the same stimuli as the degradable bead or the twodegradable species can respond to different stimuli. For example, abarcode sequence can be attached, via a disulfide bond, to apolyacrylamide bead comprising cystamine. Upon exposure of thebarcoded-bead to a reducing agent, the bead degrades and the barcodesequence is released upon breakage of both the disulfide linkage betweenthe barcode sequence and the bead and the disulfide linkages of thecystamine in the bead.

Any suitable number of species (e.g., primer, barcoded oligonucleotide)can be associated with a bead such that, upon release from the bead, thespecies (e.g., primer, e.g., barcoded oligonucleotide) are present inthe partition at a pre-defined concentration. Such pre-definedconcentration can be selected to facilitate certain reactions forgenerating a sequencing library, e.g., amplification, within thepartition. In some cases, the pre-defined concentration of the primercan be limited by the process of producing nucleic acid molecule (e.g.,oligonucleotide) bearing beads.

As will be appreciated from the above description, while referred to asdegradation of a bead, in many embodiments, degradation can refer to thedisassociation of a bound or entrained species from a bead, both withand without structurally degrading the physical bead itself. Forexample, entrained species can be released from beads through osmoticpressure differences due to, for example, changing chemicalenvironments. By way of example, alteration of bead pore volumes due toosmotic pressure differences can generally occur without structuraldegradation of the bead itself. In some cases, an increase in porevolume due to osmotic swelling of a bead can permit the release ofentrained species within the bead. In some embodiments, osmoticshrinking of a bead can cause a bead to better retain an entrainedspecies due to pore volume contraction.

Numerous chemical triggers can be used to trigger the degradation ofbeads within partitions. Examples of these chemical changes can include,but are not limited to pH-mediated changes to the integrity of acomponent within the bead, degradation of a component of a bead viacleavage of cross-linked bonds, and depolymerization of a component of abead.

In certain embodiments, a change in pH of a solution, such as anincrease in pH, can trigger degradation of a bead. In other embodiments,exposure to an aqueous solution, such as water, can trigger hydrolyticdegradation, and thus degradation of the bead. In some cases, anycombination of stimuli can trigger degradation of a bead. For example, achange in pH can enable a chemical agent (e.g., DTT) to become aneffective reducing agent.

Beads can also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat can cause melting of a bead such that a portion of thebead degrades. In other cases, heat can increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat canalso act upon heat-sensitive polymers used as materials to constructbeads.

In addition to beads and analytes, partitions that are formed caninclude a variety of different reagents and species. For example, whenlysis reagents are present within the partitions, the lysis reagents canfacilitate the release of analytes within the partition. Examples oflysis agents include bioactive reagents, such as lysis enzymes that areused for lysis of different cell types, e.g., gram positive or negativebacteria, plants, yeast, mammalian, etc., such as lysozymes,achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and avariety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc.(St. Louis, Mo.), as well as other commercially available lysis enzymes.Other lysis agents can additionally or alternatively be co-partitionedto cause the release analytes into the partitions. For example, in somecases, surfactant-based lysis solutions can be used to lyse cells,although these can be less desirable for emulsion based systems wherethe surfactants can interfere with stable emulsions. In someembodiments, lysis solutions can include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some embodiments, lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic ormechanical cellular disruption can also be used in certain embodiments,e.g., non-emulsion based partitioning such as encapsulation of analytesthat can be in addition to or in place of droplet partitioning, whereany pore size of the encapsulate is sufficiently small to retain nucleicacid fragments of a given volume, following cellular disruption.

Examples of other species that can be co-partitioned with analytes inthe partitions include, but are not limited to, DNase and RNaseinactivating agents or inhibitors, such as proteinase K, chelatingagents, such as EDTA, and other reagents employed in removing orotherwise reducing negative activity or impact of different cell lysatecomponents on subsequent processing of nucleic acids. Additionalreagents can also be co-partitioned, including endonucleases to fragmentDNA, DNA polymerase enzymes and dNTPs used to amplify nucleic acidfragments and to attach the barcode molecular tags to the amplifiedfragments.

Additional reagents can also include reverse transcriptase enzymes,including enzymes with terminal transferase activity, primers andoligonucleotides, and switch oligonucleotides (also referred to hereinas “switch oligos” or “template switching oligonucleotides”) which canbe used for template switching. In some embodiments, template switchingcan be used to increase the length of a cDNA. Template switching can beused to append a predefined nucleic acid sequence to the cDNA. In anexample of template switching, cDNA can be generated from reversetranscription of a template, e.g., cellular mRNA, where a reversetranscriptase with terminal transferase activity can add additionalnucleotides, e.g., poly(C), to the cDNA in a template independentmanner. Switch oligos can include sequences complementary to theadditional nucleotides, e.g., poly(G). The additional nucleotides (e.g.,poly(C)) on the cDNA can hybridize to the additional nucleotides (e.g.,poly(G)) on the switch oligo, whereby the switch oligo can be used bythe reverse transcriptase as template to further extend the cDNA.

Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target. In some cases, the hybridizationregion includes a series of G bases to complement the overhanging Cbases at the 3′ end of a cDNA molecule. The series of G bases caninclude 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or morethan 5 G bases. The template sequence can include any sequence to beincorporated into the cDNA. In some cases, the template region includesat least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/orfunctional sequences. Switch oligos can include deoxyribonucleic acids;ribonucleic acids; bridged nucleic acids, modified nucleic acidsincluding 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT,5-Methyl dC, 2′-deoxylnosine, Super T (5-hydroxybutynl-2′-deoxyuridine),Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlockednucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC,2′ Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), andcombinations of the foregoing.

In some embodiments, beads that are partitioned with the analyte caninclude different types of oligonucleotides bound to the bead, where thedifferent types of oligonucleotides bind to different types of analytes.For example, a bead can include one or more first oligonucleotides(which can be capture probes, for example) that can bind or hybridize toa first type of analyte, such as mRNA for example, and one or moresecond oligonucleotides (which can be capture probes, for example) thatcan bind or hybridize to a second type of analyte, such as gDNA forexample. Partitions can also include lysis agents that aid in releasingnucleic acids from the co-partitioned cell, and can also include anagent (e.g., a reducing agent) that can degrade the bead and/or breakcovalent linkages between the oligonucleotides and the bead, releasingthe oligonucleotides into the partition. The released barcodedoligonucleotides (which can also be barcoded) can hybridize with mRNAreleased from the cell and also with gDNA released from the cell.

Barcoded constructs thus formed from hybridization can include a firsttype of construct that includes a sequence corresponding to an originalbarcode sequence from the bead and a sequence corresponding to atranscript from the cell, and a second type of construct that includes asequence corresponding to the original barcode sequence from the beadand a sequence corresponding to genomic DNA from the cell. The barcodedconstructs can then be released/removed from the partition and, in someembodiments, further processed to add any additional sequences. Theresulting constructs can then be sequenced, the sequencing dataprocessed, and the results used to spatially characterize the mRNA andthe gDNA from the cell.

In another example, a partition includes a bead that includes a firsttype of oligonucleotide (e.g., a first capture probe) with a firstbarcode sequence, a poly(T) priming sequence that can hybridize with thepoly(A) tail of an mRNA transcript, and a UMI barcode sequence that canuniquely identify a given transcript. The bead also includes a secondtype of oligonucleotide (e.g., a second capture probe) with a secondbarcode sequence, a targeted priming sequence that is capable ofspecifically hybridizing with a third barcoded oligonucleotide (e.g., ananalyte capture agent) coupled to an antibody that is bound to thesurface of the partitioned cell. The third barcoded oligonucleotideincludes a UMI barcode sequence that uniquely identifies the antibody(and thus, the particular cell surface feature to which it is bound).

In this example, the first and second barcoded oligonucleotides includethe same spatial barcode sequence (e.g., the first and second barcodesequences are the same), which permits downstream association ofbarcoded nucleic acids with the partition. In some embodiments, however,the first and second barcode sequences are different.

The partition also includes lysis agents that aid in releasing nucleicacids from the cell and can also include an agent (e.g., a reducingagent) that can degrade the bead and/or break a covalent linkage betweenthe barcoded oligonucleotides and the bead, releasing them into thepartition. The first type of released barcoded oligonucleotide canhybridize with mRNA released from the cell and the second type ofreleased barcoded oligonucleotide can hybridize with the third type ofbarcoded oligonucleotide, forming barcoded constructs.

The first type of barcoded construct includes a spatial barcode sequencecorresponding to the first barcode sequence from the bead and a sequencecorresponding to the UMI barcode sequence from the first type ofoligonucleotide, which identifies cell transcripts. The second type ofbarcoded construct includes a spatial barcode sequence corresponding tothe second barcode sequence from the second type of oligonucleotide, anda UMI barcode sequence corresponding to the third type ofoligonucleotide (e.g., the analyte capture agent) and used to identifythe cell surface feature. The barcoded constructs can then bereleased/removed from the partition and, in some embodiments, furtherprocessed to add any additional sequences. The resulting constructs arethen sequenced, sequencing data processed, and the results used tocharacterize the mRNA and cell surface feature of the cell.

The foregoing discussion involves two specific examples of beads witholigonucleotides for analyzing two different analytes within apartition. More generally, beads that are partitioned can have any ofthe structures described previously, and can include any of thedescribed combinations of oligonucleotides for analysis of two or more(e.g., three or more, four or more, five or more, six or more, eight ormore, ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 ormore, 40 or more, 50 or more) different types of analytes within apartition. Examples of beads with combinations of different types ofoligonucleotides (e.g., capture probes) for concurrently analyzingdifferent combinations of analytes within partitions include, but arenot limited to: (a) genomic DNA and cell surface features (e.g., usingthe analyte capture agents described herein); (b) mRNA and a lineagetracing construct; (c) mRNA and cell methylation status; (d) mRNA andaccessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq); (e)mRNA and cell surface or intracellular proteins and/or metabolites; (f)a barcoded analyte capture agent (e.g., the MHC multimers describedherein) and a V(D)J sequence of an immune cell receptor (e.g., T-cellreceptor); and (g) mRNA and a perturbation agent (e.g., a CRISPRcrRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein). In some embodiments, aperturbation agent can be a small molecule, an antibody, a drug, anaptamer, a miRNA, a physical environmental (e.g., temperature change),or any other known perturbation agents.

Additionally, in some embodiments, the unaggregated cell ordisaggregated cells introduced and processed within partitions ordroplets as described herein, can be removed from the partition,contacted with a spatial array, and spatially-barcoded according tomethods described herein. For example, single cells of an unaggregatedcell sample can be partitioned into partitions or droplets as describedherein. The partitions or droplets can include reagents to permeabilizea cell, barcode targeted cellular analyte(s) with a cellular barcode,and amplify the barcoded analytes. The partitions or droplets can becontacted with any of the spatial arrays described herein. In someembodiments, the partition can be dissolved, such that the contents ofthe partition are placed in contact with the capture probes of thespatial array. The capture probes of the spatial array can then capturetarget analytes from the ruptured partitions or the droplets, andprocessed by the spatial workflows described herein.

(g) Analysis of Captured Analytes

(i) Sample Removal from an Array

In some embodiments, after contacting a biological sample with asubstrate that includes capture probes, a removal step can optionally beperformed to remove all or a portion of the biological sample from thesubstrate. In some embodiments, the removal step includes enzymaticand/or chemical degradation of cells of the biological sample. Forexample, the removal step can include treating the biological samplewith an enzyme (e.g., a proteinase, e.g., proteinase K) to remove atleast a portion of the biological sample from the substrate. In someembodiments, the removal step can include ablation of the tissue (e.g.,laser ablation).

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample), the method comprising: (a) optionally stainingand/or imaging a biological sample on a substrate; (b) permeabilizing(e.g., providing a solution comprising a permeabilization reagent to)the biological sample on the substrate; (c) contacting the biologicalsample with an array comprising a plurality of capture probes, wherein acapture probe of the plurality captures the biological analyte; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte; wherein the biological sample is fully orpartially removed from the substrate.

In some embodiments, a biological sample is not removed from thesubstrate. For example, the biological sample is not removed from thesubstrate prior to releasing a capture probe (e.g., a capture probebound to an analyte) from the substrate. In some embodiments, suchreleasing comprises cleavage of the capture probe from the substrate(e.g., via a cleavage domain). In some embodiments, such releasing doesnot comprise releasing the capture probe from the substrate (e.g., acopy of the capture probe bound to an analyte can be made and the copycan be released from the substrate, e.g., via denaturation). In someembodiments, the biological sample is not removed from the substrateprior to analysis of an analyte bound to a capture probe after it isreleased from the substrate. In some embodiments, the biological sampleremains on the substrate during removal of a capture probe from thesubstrate and/or analysis of an analyte bound to the capture probe afterit is released from the substrate. In some embodiments, the biologicalsample remains on the substrate during removal (e.g., via denaturation)of a copy of the capture probe (e.g., complement). In some embodiments,analysis of an analyte bound to capture probe from the substrate can beperformed without subjecting the biological sample to enzymatic and/orchemical degradation of the cells (e.g., permeabilized cells) orablation of the tissue (e.g., laser ablation).

In some embodiments, at least a portion of the biological sample is notremoved from the substrate. For example, a portion of the biologicalsample can remain on the substrate prior to releasing a capture probe(e.g., a capture prove bound to an analyte) from the substrate and/oranalyzing an analyte bound to a capture probe released from thesubstrate. In some embodiments, at least a portion of the biologicalsample is not subjected to enzymatic and/or chemical degradation of thecells (e.g., permeabilized cells) or ablation of the tissue (e.g., laserablation) prior to analysis of an analyte bound to a capture probe fromthe substrate.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample) that include: (a) optionally staining and/or imaginga biological sample on a substrate; (b) permeabilizing (e.g., providinga solution comprising a permeabilization reagent to) the biologicalsample on the substrate; (c) contacting the biological sample with anarray comprising a plurality of capture probes, wherein a capture probeof the plurality captures the biological analyte; and (d) analyzing thecaptured biological analyte, thereby spatially detecting the biologicalanalyte; where the biological sample is not removed from the substrate.

In some embodiments, provided herein are methods for spatially detectinga biological analyte of interest from a biological sample that include:(a) staining and imaging a biological sample on a substrate; (b)providing a solution comprising a permeabilization reagent to thebiological sample on the substrate; (c) contacting the biological samplewith an array on a substrate, wherein the array comprises one or morecapture probe pluralities thereby allowing the one or more pluralitiesof capture probes to capture the biological analyte of interest; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte of interest; where the biological sample is notremoved from the substrate.

In some embodiments, the method further includes selecting a region ofinterest in the biological sample to subject to spatial transcriptomicanalysis. In some embodiments, one or more of the one or more captureprobes include a capture domain. In some embodiments, one or more of theone or more capture probe pluralities comprise a unique molecularidentifier (UMI). In some embodiments, one or more of the one or morecapture probe pluralities comprise a cleavage domain. In someembodiments, the cleavage domain comprises a sequence recognized andcleaved by a uracil-DNA glycosylase, apurinic/apyrimidinic (AP)endonuclease (APE1), U uracil-specific excision reagent (USER), and/oran endonuclease VIII. In some embodiments, one or more capture probes donot comprise a cleavage domain and is not cleaved from the array.

(ii) Extended Capture Probes

In some embodiments, a capture probe can be extended (an “extendedcapture probe,” e.g., as described herein (e.g., Section II(b)(vii))).For example, extending a capture probe can include generating cDNA froma captured (hybridized) RNA. This process involves synthesis of acomplementary strand of the hybridized nucleic acid, e.g., generatingcDNA based on the captured RNA template (the RNA hybridized to thecapture domain of the capture probe). Thus, in an initial step ofextending a capture probe, e.g., the cDNA generation, the captured(hybridized) nucleic acid, e.g., RNA, acts as a template for theextension, e.g., reverse transcription, step.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, a capture domain of a capture probe includes aprimer for producing the complementary strand of a nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA (e.g., cDNA) molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if a nucleicacid (e.g., RNA) was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from Lucigen,Middleton, Wis.). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended capture probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9° N™ DNAligase, New England Biolabs), Ampligase™ (available from Lucigen,Middleton, Wis.), and SplintR (available from New England Biolabs,Ipswich, Mass.). In some embodiments, a polynucleotide tail, e.g., apoly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g., sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to a substrate specific for the affinity group. Insome embodiments, the substrate can include an antibody or antibodyfragment. In some embodiments, the substrate includes avidin orstreptavidin and the affinity group includes biotin. In someembodiments, the substrate includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the substrateincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the surface of thesubstrate, insofar as copies of the extended probes are not immobilizedon the substrate.

In some embodiments, the extended capture probe or complement oramplicon thereof is released. The step of releasing the extended captureprobe or complement or amplicon thereof from the surface of thesubstrate can be achieved in a number of ways. In some embodiments, anextended capture probe or a complement thereof is released from thearray by nucleic acid cleavage and/or by denaturation (e.g., by heatingto denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the surface of the substrate (e.g.,array) by physical means. For example, where the extended capture probeis indirectly immobilized on the array substrate, e.g., viahybridization to a surface probe, it can be sufficient to disrupt theinteraction between the extended capture probe and the surface probe.Methods for disrupting the interaction between nucleic acid moleculesinclude denaturing double stranded nucleic acid molecules are known inthe art. A straightforward method for releasing the DNA molecules (i.e.,of stripping the array of extended probes) is to use a solution thatinterferes with the hydrogen bonds of the double stranded molecules. Insome embodiments, the extended capture probe is released by a applyingheated solution, such as water or buffer, of at least 85° C., e.g., atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C. In some embodiments,a solution including salts, surfactants, etc. that can furtherdestabilize the interaction between the nucleic acid molecules is addedto release the extended capture probe from the substrate.

In some embodiments, where the extended capture probe includes acleavage domain, the extended capture probe is released from the surfaceof the substrate by cleavage. For example, the cleavage domain of theextended capture probe can be cleaved by any of the methods describedherein. In some embodiments, the extended capture probe is released fromthe surface of the substrate, e.g., via cleavage of a cleavage domain inthe extended capture probe, prior to the step of amplifying the extendedcapture probe.

In some embodiments, probes complementary to the extended capture probecan be contacted with the substrate. In some embodiments, the biologicalsample can be in contact with the substrate when the probes arecontacted with the substrate. In some embodiments, the biological samplecan be removed from the substrate prior to contacting the substrate withprobes. In some embodiments, the probes can be labeled with a detectablelabel (e.g., any of the detectable labels described herein). In someembodiments, probes that do not specially bind (e.g., hybridize) to anextended capture probe can be washed away. In some embodiments, probescomplementary to the extended capture probe can be detected on thesubstrate (e.g., imaging, any of the detection methods describedherein).

In some embodiments, probes complementary to an extended capture probecan be about 4 nucleotides to about 100 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended capture probe can be about 10 nucleotides to about 90nucleotides long. In some embodiments, probes (e.g., detectable probes)complementary to an extended capture probe can be about 20 nucleotidesto about 80 nucleotides long. In some embodiments, probes (e.g.,detectable probes) complementary to an extended capture probe can beabout 30 nucleotides to about 60 nucleotides long. In some embodiments,probes (e.g., detectable probes) complementary to an extended captureprobe can be about 40 nucleotides to about 50 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended capture probe can be about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, about 35, about 36, about 37,about 38, about 39, about 40, about 41, about 42, about 43, about 44,about 45, about 46, about 47, about 48, about 49, about 50, about 51,about 52, about 53, about 54, about 55, about 56, about 57, about 58,about 59, about 60, about 61, about 62, about 63, about 64, about 65,about 66, about 67, about 68, about 69, about 70, about 71, about 72,about 73, about 74, about 75, about 76, about 77, about 78, about 79,about 80, about 81, about 82, about 83, about 84, about 85, about 86,about 87, about 88, about 89, about 90, about 91, about 92, about 93,about 94, about 95, about 96, about 97, about 98, and about 99nucleotides long.

In some embodiments, about 1 to about 100 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe. In some embodiments, about 1 to about 10 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedcapture probe. In some embodiments, about 10 to about 100 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended capture probe. In some embodiments, about 20 to about 90 probescan be contacted to the substrate and specifically bind (e.g.,hybridize) to an extended capture probe. In some embodiments, about 30to about 80 probes (e.g., detectable probes) can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe. In some embodiments, about 40 to about 70 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedcapture probe. In some embodiments, about 50 to about 60 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended capture probe. In some embodiments, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, about 45, about 46, about47, about 48, about 49, about 50, about 51, about 52, about 53, about54, about 55, about 56, about 57, about 58, about 59, about 60, about61, about 62, about 63, about 64, about 65, about 66, about 67, about68, about 69, about 70, about 71, about 72, about 73, about 74, about75, about 76, about 77, about 78, about 79, about 80, about 81, about82, about 83, about 84, about 85, about 86, about 87, about 88, about89, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, and about 99 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe.

In some embodiments, the probes can be complementary to a single analyte(e.g., a single gene). In some embodiments, the probes can becomplementary to one or more analytes (e.g., analytes in a family ofgenes). In some embodiments, the probes (e.g., detectable probes) can befor a panel of genes associated with a disease (e.g., cancer,Alzheimer's disease, Parkinson's disease).

(iii) Cleavage Domain

Capture probes can optionally include a “cleavage domain,” where one ormore segments or regions of the capture probe (e.g., spatial barcodesand/or UMIs) can be releasably, cleavably, or reversibly attached to afeature, or some other substrate, so that spatial barcodes and/or UMIscan be released or be releasable through cleavage of a linkage betweenthe capture probe and the feature, or released through degradation ofthe underlying substrate or chemical substrate, allowing the spatialbarcode(s) and/or UMI(s) of the cleaved capture probe to be accessed orbe accessible by other reagents, or both. Non-limiting aspects ofcleavage domains are described herein (e.g., in Section II(b)(ii)).

In some embodiments, the capture probe is linked, (e.g., via a disulfidebond), to a feature. In some embodiments, the capture probe is linked toa feature via a propylene group (e.g., Spacer C3). A reducing agent canbe added to break the various disulfide bonds, resulting in release ofthe capture probe including the spatial barcode sequence. In anotherexample, heating can also result in degradation and release of theattached capture probe. In some embodiments, the heating is done bylaser (e.g., laser ablation) and features at specific locations can bedegraded. In addition to thermally cleavable bonds, disulfide bonds,photo-sensitive bonds, and UV sensitive bonds, other non-limitingexamples of labile bonds that can be coupled to a capture probe (e.g.,spatial barcode) include an ester linkage (e.g., cleavable with an acid,a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable viasodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), asulfone linkage (e.g., cleavable via a base), a silyl ether linkage(e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable viaan amylase), a peptide linkage (e.g., cleavable via a protease), or aphosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)).

In some embodiments, the cleavage domain includes a poly(U) sequencewhich can be cleaved by a mixture of Uracil DNA glycosylase (UDG) andthe DNA glycosylase-lyase Endonuclease VIII, commercially known as theUSER™ enzyme. In some embodiments, the cleavage domain can be a singleU. In some embodiments, the cleavage domain can be an abasic site thatcan be cleaved with an abasic site-specific endonuclease (e.g.,Endonuclease IV or Endonuclease VIII).

In some embodiments, the cleavage domain of the capture probe is anucleotide sequence within the capture probe that is cleavedspecifically, e.g., physically by light or heat, chemically orenzymatically. The location of the cleavage domain within the captureprobe will depend on whether or not the capture probe is immobilized onthe substrate such that it has a free 3′ end capable of functioning asan extension primer (e.g., by its 5′ or 3′ end). For example, if thecapture probe is immobilized by its 5′ end, the cleavage domain will belocated 5′ to the spatial barcode and/or UMI, and cleavage of saiddomain results in the release of part of the capture probe including thespatial barcode and/or UMI and the sequence 3′ to the spatial barcode,and optionally part of the cleavage domain, from a feature.Alternatively, if the capture probe is immobilized by its 3′ end, thecleavage domain will be located 3′ to the capture domain (and spatialbarcode) and cleavage of said domain results in the release of part ofthe capture probe including the spatial barcode and the sequence 3′ tothe spatial barcode from a feature. In some embodiments, cleavageresults in partial removal of the cleavage domain. In some embodiments,cleavage results in complete removal of the cleavage domain,particularly when the capture probes are immobilized via their 3′ end asthe presence of a part of the cleavage domain can interfere with thehybridization of the capture domain and the target nucleic acid and/orits subsequent extension.

(iv) Sequencing

After analytes from the sample have hybridized or otherwise beenassociated with capture probes, analyte capture agents, or otherbarcoded oligonucleotide sequences according to any of the methodsdescribed above in connection with the general spatial cell-basedanalytical methodology, the barcoded constructs that result fromhybridization/association are analyzed via sequencing to identify theanalytes.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample. Alternatively, if the barcoded sample has been separated intofragments, cell groups, or individual cells, as described above,sequencing can be performed on individual fragments, cell groups, orcells. For analytes that have been barcoded via partitioning with beads,as described above, individual analytes (e.g., cells, or cellularcontents following lysis of cells) can be extracted from the partitionsby breaking the partitions, and then analyzed by sequencing to identifythe analytes.

A wide variety of different sequencing methods can be used to analyzebarcoded analyte constructs. In general, sequenced polynucleotides canbe, for example, nucleic acid molecules such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA), including variants or derivativesthereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acidmolecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various commercialsystems. More generally, sequencing can be performed using nucleic acidamplification, polymerase chain reaction (PCR) (e.g., digital PCR anddroplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplexPCR, PCR-based singleplex methods, emulsion PCR), and/or isothermalamplification.

Other examples of methods for sequencing genetic material include, butare not limited to, DNA hybridization methods (e.g., Southern blotting),restriction enzyme digestion methods, Sanger sequencing methods,next-generation sequencing methods (e.g., single-molecule real-timesequencing, nanopore sequencing, and Polony sequencing), ligationmethods, and microarray methods. Additional examples of sequencingmethods that can be used include targeted sequencing, single moleculereal-time sequencing, exon sequencing, electron microscopy-basedsequencing, panel sequencing, transistor-mediated sequencing, directsequencing, random shotgun sequencing, Sanger dideoxy terminationsequencing, whole-genome sequencing, sequencing by hybridization,pyrosequencing, capillary electrophoresis, gel electrophoresis, duplexsequencing, cycle sequencing, single-base extension sequencing,solid-phase sequencing, high-throughput sequencing, massively parallelsignature sequencing, co-amplification at lower denaturationtemperature-PCR (COLD-PCR), sequencing by reversible dye terminator,paired-end sequencing, near-term sequencing, exonuclease sequencing,sequencing by ligation, short-read sequencing, single-moleculesequencing, sequencing-by-synthesis, real-time sequencing,reverse-terminator sequencing, nanopore sequencing, MS-PET sequencing,and any combinations thereof.

Sequence analysis of the nucleic acid molecules (including barcodednucleic acid molecules or derivatives thereof) can be direct orindirect. Thus, the sequence analysis substrate (which can be viewed asthe molecule which is subjected to the sequence analysis step orprocess) can be the barcoded nucleic acid molecule or it can be amolecule which is derived therefrom (e.g., a complement thereof). Thus,for example, in the sequence analysis step of a sequencing reaction, thesequencing template can be the barcoded nucleic acid molecule or it canbe a molecule derived therefrom. For example, a first and/or secondstrand DNA molecule can be directly subjected to sequence analysis(e.g., sequencing), i.e., can directly take part in the sequenceanalysis reaction or process (e.g., the sequencing reaction orsequencing process, or be the molecule which is sequenced or otherwiseidentified). Alternatively, the barcoded nucleic acid molecule can besubjected to a step of second strand synthesis or amplification beforesequence analysis (e.g., sequencing or identification by anothertechnique). The sequence analysis substrate (e.g., template) can thus bean amplicon or a second strand of a barcoded nucleic acid molecule.

In some embodiments, both strands of a double stranded molecule can besubjected to sequence analysis (e.g., sequenced). In some embodiments,single stranded molecules (e.g., barcoded nucleic acid molecules) can beanalyzed (e.g., sequenced). To perform single molecule sequencing, thenucleic acid strand can be modified at the 3′ end.

Massively parallel pyrosequencing techniques can be used for sequencingnucleic acids. In pyrosequencing, the nucleic acid is amplified insidewater droplets in an oil solution (emulsion PCR), with each dropletcontaining a single nucleic acid template attached to a singleprimer-coated bead that then forms a clonal colony. The sequencingsystem contains many picolitre-volume wells each containing a singlebead and sequencing enzymes. Pyrosequencing uses luciferase to generatelight for detection of the individual nucleotides added to the nascentnucleic acid and the combined data are used to generate sequence reads.

As another example application of pyrosequencing, released PPi can bedetected by being immediately converted to adenosine triphosphate (ATP)by ATP sulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons, such as described in Ronaghi, et al., Anal.Biochem. 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001);Ronaghi et al. Science 281 (5375), 363 (1998); and U.S. Pat. Nos.6,210,891, 6,258,568, and 6,274,320, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, a massively parallel sequencing technique can bebased on reversible dye-terminators. As an example, DNA molecules arefirst attached to primers on, e.g., a glass or silicon substrate, andamplified so that local clonal colonies are formed (bridgeamplification). Four types of ddNTPs are added, and non-incorporatednucleotides are washed away. Unlike pyrosequencing, the DNA is onlyextended one nucleotide at a time due to a blocking group (e.g., 3′blocking group present on the sugar moiety of the ddNTP). A detectoracquires images of the fluorescently labelled nucleotides, and then thedye along with the terminal 3′ blocking group is chemically removed fromthe DNA, as a precursor to a subsequent cycle. This process can berepeated until the required sequence data is obtained.

In some embodiments, sequencing is performed by detection of hydrogenions that are released during the polymerization of DNA. A microwellcontaining a template DNA strand to be sequenced can be flooded with asingle type of nucleotide. If the introduced nucleotide is complementaryto the leading template nucleotide, it is incorporated into the growingcomplementary strand. This causes the release of a hydrogen ion thattriggers a hypersensitive ion sensor, which indicates that a reactionhas occurred. If homopolymer repeats are present in the templatesequence, multiple nucleotides will be incorporated in a single cycle.This leads to a corresponding number of released hydrogen ions and aproportionally higher electronic signal.

In some embodiments, sequencing can be performed in situ. In situsequencing methods are particularly useful, for example, when thebiological sample remains intact after analytes on the sample surface(e.g., cell surface analytes) or within the sample (e.g., intracellularanalytes) have been barcoded. In situ sequencing typically involvesincorporation of a labeled nucleotide (e.g., fluorescently labeledmononucleotides or dinucleotides) in a sequential, template-dependentmanner or hybridization of a labeled primer (e.g., a labeled randomhexamer) to a nucleic acid template such that the identities (i.e.,nucleotide sequence) of the incorporated nucleotides or labeled primerextension products can be determined, and consequently, the nucleotidesequence of the corresponding template nucleic acid. Aspects of in situsequencing are described, for example, in Mitra et al., (2003)Anal.Biochem. 320, 55-65, and Lee et al., (2014) Science, 343(6177),1360-1363, the entire contents of each of which are incorporated hereinby reference.

In addition, examples of methods and systems for performing in situsequencing are described in PCT Patent Application Publication Nos.WO2014/163886, WO2018/045181, WO2018/045186, and in U.S. Pat. Nos.10,138,509 and 10,179,932, the entire contents of each of which areincorporated herein by reference. Exemplary techniques for in situsequencing include, but are not limited to, STARmap (described forexample in Wang et al., (2018) Science, 361(6499) 5691), MERFISH(described for example in Moffitt, (2016) Methods in Enzymology, 572,1-49), and FISSEQ (described for example in U.S. Patent ApplicationPublication No. 2019/0032121). The entire contents of each of theforegoing references are incorporated herein by reference.

For analytes that have been barcoded via partitioning, barcoded nucleicacid molecules or derivatives thereof (e.g., barcoded nucleic acidmolecules to which one or more functional sequences have been added, orfrom which one or more features have been removed) can be pooled andprocessed together for subsequent analysis such as sequencing on highthroughput sequencers. Processing with pooling can be implemented usingbarcode sequences. For example, barcoded nucleic acid molecules of agiven partition can have the same barcode, which is different frombarcodes of other spatial partitions. Alternatively, barcoded nucleicacid molecules of different partitions can be processed separately forsubsequent analysis (e.g., sequencing).

In some embodiments, where capture probes do not contain a spatialbarcode, the spatial barcode can be added after the capture probecaptures analytes from a biological sample and before analysis of theanalytes. When a spatial barcode is added after an analyte is captured,the barcode can be added after amplification of the analyte (e.g.,reverse transcription and polymerase amplification of RNA). In someembodiments, analyte analysis uses direct sequencing of one or morecaptured analytes, such as direct sequencing of hybridized RNA. In someembodiments, direct sequencing is performed after reverse transcriptionof hybridized RNA. In some embodiments direct sequencing is performedafter amplification of reverse transcription of hybridized RNA.

In some embodiments, direct sequencing of captured RNA is performed bysequencing-by-synthesis (SBS). In some embodiments, a sequencing primeris complementary to a sequence in one or more of the domains of acapture probe (e.g., functional domain). In such embodiments,sequencing-by-synthesis can include reverse transcription and/oramplification in order to generate a template sequence (e.g., functionaldomain) from which a primer sequence can bind.

SBS can involve hybridizing an appropriate primer, sometimes referred toas a sequencing primer, with the nucleic acid template to be sequenced,extending the primer, and detecting the nucleotides used to extend theprimer. Preferably, the nucleic acid used to extend the primer isdetected before a further nucleotide is added to the growing nucleicacid chain, thus allowing base-by-base in situ nucleic acid sequencing.The detection of incorporated nucleotides is facilitated by includingone or more labelled nucleotides in the primer extension reaction. Toallow the hybridization of an appropriate sequencing primer to thenucleic acid template to be sequenced, the nucleic acid template shouldnormally be in a single stranded form. If the nucleic acid templatesmaking up the nucleic acid features are present in a double strandedform these can be processed to provide single stranded nucleic acidtemplates using methods well known in the art, for example bydenaturation, cleavage, etc. The sequencing primers which are hybridizedto the nucleic acid template and used for primer extension arepreferably short oligonucleotides, for example, 15 to 25 nucleotides inlength. The sequencing primers can be provided in solution or in animmobilized form. Once the sequencing primer has been annealed to thenucleic acid template to be sequenced by subjecting the nucleic acidtemplate and sequencing primer to appropriate conditions, primerextension is carried out, for example using a nucleic acid polymeraseand a supply of nucleotides, at least some of which are provided in alabelled form, and conditions suitable for primer extension if asuitable nucleotide is provided.

Preferably after each primer extension step, a washing step is includedin order to remove unincorporated nucleotides which can interfere withsubsequent steps. Once the primer extension step has been carried out,the nucleic acid colony is monitored to determine whether a labellednucleotide has been incorporated into an extended primer. The primerextension step can then be repeated to determine the next and subsequentnucleotides incorporated into an extended primer. If the sequence beingdetermined is unknown, the nucleotides applied to a given colony areusually applied in a chosen order which is then repeated throughout theanalysis, for example dATP, dTTP, dCTP, dGTP.

SBS techniques which can be used are described for example, but notlimited to, those in U.S. Patent App. Pub. No. 2007/0166705, U.S. PatentApp. Pub. No. 2006/0188901, U.S. Pat. No. 7,057,026, U.S. Patent App.Pub. No. 2006/0240439, U.S. Patent App. Pub. No. 2006/0281109, PCTPatent App. Pub. No. WO 05/065814, U.S. Patent App. Pub. No.2005/0100900, PCT Patent App. Pub. No. WO 06/064199, PCT Patent App.Pub. No. WO07/010,251, U.S. Patent App. Pub. No. 2012/0270305, U.S.Patent App. Pub. No. 2013/0260372, and U.S. Patent App. Pub. No.2013/0079232, the entire contents of each of which are incorporatedherein by reference.

In some embodiments, direct sequencing of captured RNA is performed bysequential fluorescence hybridization (e.g., sequencing byhybridization). In some embodiments, a hybridization reaction where RNAis hybridized to a capture probe is performed in situ. In someembodiments, captured RNA is not amplified prior to hybridization with asequencing probe. In some embodiments, RNA is amplified prior tohybridization with sequencing probes (e.g., reverse transcription tocDNA and amplification of cDNA). In some embodiments, amplification isperformed using single-molecule hybridization chain reaction. In someembodiments, amplification is performed using rolling chainamplification.

Sequential fluorescence hybridization can involve sequentialhybridization of probes including degenerate primer sequences and adetectable label. A degenerate primer sequence is a shortoligonucleotide sequence which is capable of hybridizing to any nucleicacid fragment independent of the sequence of said nucleic acid fragment.For example, such a method could include the steps of: (a) providing amixture including four probes, each of which includes either A, C, G, orT at the 5′-terminus, further including degenerate nucleotide sequenceof 5 to 11 nucleotides in length, and further including a functionaldomain (e.g., fluorescent molecule) that is distinct for probes with A,C, G, or T at the 5′-terminus; (b) associating the probes of step (a) tothe target polynucleotide sequences, whose sequence needs will bedetermined by this method; (c) measuring the activities of the fourfunctional domains and recording the relative spatial location of theactivities; (d) removing the reagents from steps (a)-(b) from the targetpolynucleotide sequences; and repeating steps (a)-(d) for n cycles,until the nucleotide sequence of the spatial domain for each bead isdetermined, with modification that the oligonucleotides used in step (a)are complementary to part of the target polynucleotide sequences and thepositions 1 through n flanking the part of the sequences. Because thebarcode sequences are different, in some embodiments, these additionalflanking sequences are degenerate sequences. The fluorescent signal fromeach spot on the array for cycles 1 through n can be used to determinethe sequence of the target polynucleotide sequences.

In some embodiments, direct sequencing of captured RNA using sequentialfluorescence hybridization is performed in vitro. In some embodiments,captured RNA is amplified prior to hybridization with a sequencing probe(e.g., reverse transcription to cDNA and amplification of cDNA). In someembodiments, a capture probe containing captured RNA is exposed to thesequencing probe targeting coding regions of RNA. In some embodiments,one or more sequencing probes are targeted to each coding region. Insome embodiments, the sequencing probe is designed to hybridize withsequencing reagents (e.g., a dye-labeled readout oligonucleotides). Asequencing probe can then hybridize with sequencing reagents. In someembodiments, output from the sequencing reaction is imaged. In someembodiments, a specific sequence of cDNA is resolved from an image of asequencing reaction. In some embodiments, reverse transcription ofcaptured RNA is performed prior to hybridization to the sequencingprobe. In some embodiments, the sequencing probe is designed to targetcomplementary sequences of the coding regions of RNA (e.g., targetingcDNA).

In some embodiments, a captured RNA is directly sequenced using ananopore-based method. In some embodiments, direct sequencing isperformed using nanopore direct RNA sequencing in which captured RNA istranslocated through a nanopore. A nanopore current can be recorded andconverted into a base sequence. In some embodiments, captured RNAremains attached to a substrate during nanopore sequencing. In someembodiments, captured RNA is released from the substrate prior tonanopore sequencing. In some embodiments, where the analyte of interestis a protein, direct sequencing of the protein can be performed usingnanopore-based methods. Examples of nanopore-based sequencing methodsthat can be used are described in Deamer et al., Trends Biotechnol. 18,14 7-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li etal., Nat. Mater. 2:611-615 (2003); Soni et al., Clin. Chem. 53,1996-2001 (2007); Healy et al., Nanomed. 2, 459-481 (2007); Cockroft etal., J. Am. Chem. Soc. 130, 818-820 (2008); and in U.S. Pat. No.7,001,792. The entire contents of each of the foregoing references areincorporated herein by reference.

In some embodiments, direct sequencing of captured RNA is performedusing single molecule sequencing by ligation. Such techniques utilizeDNA ligase to incorporate oligonucleotides and identify theincorporation of such oligonucleotides. The oligonucleotides typicallyhave different labels that are correlated with the identity of aparticular nucleotide in a sequence to which the oligonucleotideshybridize. Aspects and features involved in sequencing by ligation aredescribed, for example, in Shendure et al. Science (2005), 309:1728-1732, and in U.S. Pat. Nos. 5,599,675; 5,750,341; 6,969,488;6,172,218; and 6,306,597, the entire contents of each of which areincorporated herein by reference.

In some embodiments, nucleic acid hybridization can be used forsequencing. These methods utilize labeled nucleic acid decoder probesthat are complementary to at least a portion of a barcode sequence.Multiplex decoding can be performed with pools of many different probeswith distinguishable labels. Non-limiting examples of nucleic acidhybridization sequencing are described for example in U.S. Pat. No.8,460,865, and in Gunderson et al., Genome Research 14:870-877 (2004),the entire contents of each of which are incorporated herein byreference.

In some embodiments, commercial high-throughput digital sequencingtechniques can be used to analyze barcode sequences, in which DNAtemplates are prepared for sequencing not one at a time, but in a bulkprocess, and where many sequences are read out preferably in parallel,or alternatively using an ultra-high throughput serial process thatitself may be parallelized. Examples of such techniques includeIllumina® sequencing (e.g., flow cell-based sequencing by synthesistechniques), using modified nucleotides (such as commercialized inHiSeq™ and additional sequencing technology instruments by Illumina,Inc., San Diego, Calif.), HeliScope™ by Helicos Biosciences Corporation,Cambridge, Mass., and PacBio RS by Pacific Biosciences of California,Inc., Menlo Park, Calif.), sequencing by ion detection technologies (IonTorrent, Inc., South San Francisco, Calif.), and sequencing of DNAnanoballs (Complete Genomics, Inc., Mountain View, Calif.).

In some embodiments, detection of a proton released upon incorporationof a nucleotide into an extension product can be used in the methodsdescribed herein. For example, the sequencing methods and systemsdescribed in U.S. Patent Application Publication Nos. 2009/0026082,2009/0127589, 2010/0137143, and 2010/0282617, can be used to directlysequence barcodes.

In some embodiments, real-time monitoring of DNA polymerase activity canbe used during sequencing. For example, nucleotide incorporations can bedetected through fluorescence resonance energy transfer (FRET), asdescribed for example in Levene et al., Science (2003), 299, 682-686,Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al.,Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181. The entire contentsof each of the foregoing references are incorporated herein by referenceherein.

(v) Temporal Analysis

In some embodiments, the methods described herein can be used to assessanalyte levels and/or expression in a cell or a biological sample overtime (e.g., before or after treatment with an agent or different stagesof differentiation). In some examples, the methods described herein canbe performed on multiple similar biological samples or cells obtainedfrom the subject at a different time points (e.g., before or aftertreatment with an agent, different stages of differentiation, differentstages of disease progression, different ages of the subject, before orafter physical perturbation, before or after treatment with aperturbation agent as described herein, or before or after developmentof resistance to an agent). As described herein, a “perturbation agent”or “perturbation reagent” can be a small molecule, an antibody, a drug,an aptamer, a nucleic acid (e.g., miRNA), a CRISPR crRNA/sgRNA, TALEN,zinc finger nuclease, antisense oligonucleotide a physical environmental(e.g., temperature change), and/or any other known perturbation agentswhere the agent alters equilibrium or homeostasis.

In some embodiments, the methods described herein can be performed onmultiple similar biological samples or cells obtained from the subjectat 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. For example, the multiplesimilar biological samples can be repetitive samples from the samesubject, the same tissue, the same organoid, the same cell suspension,or any other biological sample described herein. In some embodiments,the methods described herein can be performed on the same biologicalsample or cells obtained from the subject at a different time points(e.g., before or after treatment with a perturbation agent, differentstages of differentiation, different stages of disease progression,different ages of the subject, or before or after development ofresistance to an agent). In some embodiments, a perturbation agent canbe small-molecules, antibodies, nucleic acids, peptides, and/or otherexternal stimuli (e.g., temperature change). In some embodiments, thebiological sample is contacted with a different array at each timepoint.

In some embodiments, a sample can be placed in a controlled environmentpermissive for cellular growth and/or maintenance, and/or to preventhypoxia. In some embodiments, a controlled environment allows a sampleto be analyzed at different time points. Barcoded arrays can be placedproximal to (e.g., on top of) the sample and imaged using a microscopeor other suitable instrument to register the relative position of thebiological sample to the barcoded array, optionally using opticallyencoded fiducial markers. An electric field can be applied for a periodof time, such that biological analytes (e.g., DNA, RNA, proteins,metabolites, small molecules, lipids, and the like) are released fromthe sample and captured by capture probes on the spatially-barcodedarray, preserving spatial information of the sample. The barcoded arraycan be removed, and the spatial and molecular information therein isdetermined (e.g., by performing library construction for next generationsequencing or in situ sequencing). Sequencing can be followed bycomputational analysis to correlate the molecular information (e.g.,gene expression values with the spatial barcode). These steps can berepeated one or more times to capture the spatial information ofanalytes at different time-points.

In some embodiments, methods as described herein can be combined with acell migration assay. A cell migration assay can comprise one or moremicroprinted lines, or suspended 3D nanofibers, on which the cellsmigrate. Migration using these assays can be measured by imaging cellmigration and/or contacting migrated cells with a spatially-barcodedarray. An array used in a cell migration assay can comprise one or morechannels on the substrate of the array, e.g., to confine cell migrationto one dimension along the substrate. Additionally, the channels candirect the migration of a cell such that it does not contact anothercell on the array (e.g., the channels do not overlap with each other),and in some embodiments, the channels are about the same width as orwider than a cell (e.g., for a mammalian cell, a channel can have awidth of about 2 μm to about 10 μm). Cellular location on thespatially-barcoded array can be identified using any method describedherein.

In some embodiments, cells can be disposed on an array as describedherein and allowed to migrate. Cell migration in cell migration assayscan be used to measure target phenotypes (e.g., phenotype forinvasiveness). In some embodiments, the cell migration distance can bemeasured and correlated to a biological analyte. Reagents can be addedto the array to facilitate cell migration. For example, the array can becoated with one or more extracellular matrix (ECM) components (e.g.,basement membrane extract (BME), laminin I, collagen I, collagen IV,fibronectin, vitronectin, elastin), a cell culture medium, achemoattractant, a chemorepellant, or a combination thereof. In someembodiments, a reagent such as a chemoattractant or chemorepellant canbe disposed on only a portion of the array, present as a gradient alongthe one or more axis or channels of the array, or a combination thereof.

(vi) Spatially Resolving Analyte Information

In some embodiments, a lookup table (LUT) can be used to associate oneproperty with another property of a feature. These properties include,e.g., locations, barcodes (e.g., nucleic acid barcode molecules),spatial barcodes, optical labels, molecular tags, and other properties.

In some embodiments, a lookup table can associate a nucleic acid barcodemolecule with a feature. In some embodiments, an optical label of afeature can permit associating the feature with a biological particle(e.g., cell or nuclei). The association of a feature with a biologicalparticle can further permit associating a nucleic acid sequence of anucleic acid molecule of the biological particle to one or more physicalproperties of the biological particle (e.g., a type of a cell or alocation of the cell). For example, based on the relationship betweenthe barcode and the optical label, the optical label can be used todetermine the location of a feature, thus associating the location ofthe feature with the barcode sequence of the feature. Subsequentanalysis (e.g., sequencing) can associate the barcode sequence and theanalyte from the sample. Accordingly, based on the relationship betweenthe location and the barcode sequence, the location of the biologicalanalyte can be determined (e.g., in a specific type of cell or in a cellat a specific location of the biological sample).

In some embodiments, a feature can have a plurality of nucleic acidbarcode molecules attached thereto. The plurality of nucleic acidbarcode molecules can include barcode sequences. The plurality ofnucleic acid molecules attached to a given feature can have the samebarcode sequences, or two or more different barcode sequences. Differentbarcode sequences can be used to provide improved spatial locationaccuracy.

As discussed above, analytes obtained from a sample, such as RNA, DNA,peptides, lipids, and proteins, can be further processed. In particular,the contents of individual cells from the sample can be provided withunique spatial barcode sequences such that, upon characterization of theanalytes, the analytes can be attributed as having been derived from thesame cell. More generally, spatial barcodes can be used to attributeanalytes to corresponding spatial locations in the sample. For example,hierarchical spatial positioning of multiple pluralities of spatialbarcodes can be used to identify and characterize analytes over aparticular spatial region of the sample. In some embodiments, thespatial region corresponds to a particular spatial region of interestpreviously identified, e.g., a particular structure of cytoarchitecturepreviously identified. In some embodiments, the spatial regioncorresponds to a small structure or group of cells that cannot be seenwith the naked eye. In some embodiments, a unique molecular identifiercan be used to identify and characterize analytes at a single celllevel.

The analyte can include a nucleic acid molecule, which can be barcodedwith a barcode sequence of a nucleic acid barcode molecule. In someembodiments, the barcoded analyte can be sequenced to obtain a nucleicacid sequence. In some embodiments, the nucleic acid sequence caninclude genetic information associated with the sample. The nucleic acidsequence can include the barcode sequence, or a complement thereof. Thebarcode sequence, or a complement thereof, of the nucleic acid sequencecan be electronically associated with the property (e.g., color and/orintensity) of the analyte using the LUT to identify the associatedfeature in an array.

(vii) Proximity Capture

In some embodiments, two- or three-dimensional spatial profiling of oneor more analytes present in a biological sample can be performed using aproximity capture reaction, which is a reaction that detects twoanalytes that are spatially close to each other and/or interacting witheach other. For example, a proximity capture reaction can be used todetect sequences of DNA that are close in space to each other, e.g., theDNA sequences can be within the same chromosome, but separated by about700 bp or less. As another example, a proximity capture reaction can beused to detect protein associations, e.g., two proteins that interactwith each other. A proximity capture reaction can be performed in situto detect two analytes that are spatially close to each other and/orinteracting with each other inside a cell. Non-limiting examples ofproximity capture reactions include DNA nanoscopy, DNA microscopy, andchromosome conformation capture methods. Chromosome conformation capture(3C) and derivative experimental procedures can be used to estimate thespatial proximity between different genomic elements. Non-limitingexamples of chromatin capture methods include chromosome conformationcapture (3-C), conformation capture-on-chip (4-C), 5-C, ChIA-PET, Hi-C,targeted chromatin capture (T2C). Examples of such methods aredescribed, for example, in Miele et al., Methods Mol Biol. (2009), 464,Simonis et al., Nat. Genet. (2006), 38(11): 1348-54, Raab et al., Embo.J. (2012), 31(2): 330-350, and Eagen et al., Trends Biochem. Sci. (2018)43(6): 469-478, the entire contents of each of which is incorporatedherein by reference.

In some embodiments, the proximity capture reaction includes proximityligation. In some embodiments, proximity ligation can include usingantibodies with attached DNA strands that can participate in ligation,replication, and sequence decoding reactions. For example, a proximityligation reaction can include oligonucleotides attached to pairs ofantibodies that can be joined by ligation if the antibodies have beenbrought in proximity to each oligonucleotide, e.g., by binding the sametarget protein (complex), and the DNA ligation products that form arethen used to template PCR amplification, as described for example inSoderberg et al., Methods. (2008), 45(3): 227-32, the entire contents ofwhich are incorporated herein by reference. In some embodiments,proximity ligation can include chromosome conformation capture methods.

In some embodiments, the proximity capture reaction is performed onanalytes within about 400 nm distance (e.g., about 300 nm, about 200 nm,about 150 nm, about 100 nm, about 50 nm, about 25 nm, about 10 nm, orabout 5 nm) from each other. In general, proximity capture reactions canbe reversible or irreversible.

(viii) Feature Removal from an Array

A spatially-barcoded array can be contacted with a biological sample tospatially detect analytes present in the biological sample. In someembodiments, the features (e.g., gel pads, beads) can be removed fromthe substrate surface for additional analysis (e.g., imaging,sequencing, or quantification). For example, features on a substratedelivered by a droplet manipulation system as described herein can beremoved from the substrate surface. In some embodiments, the features(e.g., gel pads, beads) can be removed mechanically (e.g., scraping), byan enzymatic reaction, or by a chemical reaction. In some embodiments,the features (e.g., gel pads, beads) can be aspirated. In someembodiments, after the features are removed (by any method), thefeatures can be combined with a uniquely barcoded bead. In someembodiments, the oligonucleotides within a feature can be ligated orhybridized to the barcode sequence on the barcoded bead. For example,the spatial barcode oligonucleotide within a feature can be ligated tothe barcode sequence on the barcoded bead. Additionally, the captureprobes can be ligated to the barcode sequence on the barcoded bead. Insome embodiments, the features and the bead can be partitioned. In someembodiments, the features (e.g., gels pads, beads) and the uniquelybarcoded bead can be partitioned into a vesicle. In some embodiments,the vesicle can have a lipid bilayer. In some embodiments, the featuresand the bead can be encapsulated. In some embodiments, the features andthe bead can be encapsulated in an oil emulsion. In some embodiments,the features and the bead can be encapsulated in a water-in-oilemulsion. Once partitioned, the features (e.g., gel pads, beads) can beprocessed for further analysis (e.g., quantitation, amplification, orsequencing) according to any method described herein.

(ix) Other Applications

The spatial analysis methods described herein can be used to detect andcharacterize the spatial distribution of one or more haplotypes in abiological sample. As used in the present disclosure, a haplotype isused to describe one or more mutations, DNA variations, polymorphisms ina given segment of the genome, which can be used to classify the geneticsegment, or a collection of alleles or genetic segments containingsingle nucleotide polymorphisms (SNPs). Haplotype association studiesare used to inform a greater understanding of biological conditions. Forexample, identifying and characterizing haplotype variants at orassociated with putative disease loci in humans can provide a foundationfor mapping genetic causes underlying disease susceptibility. The term“locus” (plural “loci”), as used in the art, can be a fixed location ona chromosome, including the location of a gene or a genetic marker,which can contain a plurality of haplotypes, including alleles and SNPs.

Variant haplotype detection is a technique used to identify heterozygouscells in single cell studies. In combination with spatial analysis,variant haplotype detection can further provide novel information on thedistribution of heterozygous cells in biological samples (e.g., tissues)affected by or exhibiting a variety of biological conditions. These datamay reveal causal relationships between variant haplotypes and diseaseoutcomes, to aid in identification of disease-associated variants, or toreveal heterogeneity within a biological sample.

In some embodiments, variant haplotype detection is a technique that canbe used in combination with, in addition to, or as a part of, thespatial analysis methods described herein. Briefly, variant haplotypedetection can include providing inputs for executing an algorithm on acomputer system, and performing an analysis to identify and determinethe spatial distribution of haplotypes. One input can be a plurality ofsequence reads obtained from a two-dimensional spatial array in contactwith a biological sample and subsequently aligned to a genome. Thesequence reads can also contain spatial barcodes with positionalinformation, such that the sequence reads can be mapped to a location onthe biological sample. Other inputs can include electronic data files ofgene sequence variations, or haplotypes, and a reference genome. Foreach locus, the corresponding sequence reads and variant haplotypes arealigned to determine the haplotype identity of each sequence read. Thehaplotype identity and the spatial barcode of the sequence reads arethen categorized to determine the spatial distribution of haplotypeswithin the biological sample. As described above, this spatialdistribution can be used to characterize a biological condition of thesample. In some embodiments, sequence reads are obtained by in situsequencing of the two-dimensional array of positions on the substrate,while in some embodiments, sequence reads are obtained byhigh-throughput sequencing. In some embodiments, other methods forgenerating sequence reads described herein are used, such as paired endsequencings.

In some embodiments, a respective loci in the plurality of loci isbi-allelic and the corresponding set of haplotypes for the respectiveloci consists of a first allele and a second allele. In some suchembodiments, the respective loci includes a heterozygous singlenucleotide polymorphism (SNP), a heterozygous insert, or a heterozygousdeletion.

In some embodiments, analytes captured by any of the spatial analysismethods described herein can be analyzed (e.g., sequenced) via in situsequencing methods. For example, a substrate including a plurality ofcapture probes (e.g., an array), attached either directly or indirectly(e.g., via a feature), that include a spatial barcode and a capturedomain. In some embodiments, the capture domain can be configured tointeract (e.g., hybridize) with an analyte (e.g., mRNA). In someembodiments, a biological sample can be contacted to the array such thatthe capture domain of the capture probe interacts with (e.g.,hybridizes) the analyte. In some embodiments, the capture probe canfunction as a template for a hybridization or ligation reaction with thecaptured analyte. For example, a reverse transcription reaction can beperformed to extend the 3′ end of a capture probe hybridized to theanalyte using any of the exemplary reverse transcriptases describedherein, thereby generating an extended capture probe (e.g., an extendedcapture probe including the spatial barcode and a sequence that iscomplementary to a sequence in the analyte). After the extended captureprobe is synthesized, a second strand that is complementary to theextended capture probe can be synthesized. In some embodiments, secondstrand synthesis can be performed using any of the methods describedherein. In some embodiments, amine-modified nucleotides can be used whengenerating the extended capture probe or the second strand, or both. Forexample, the amine-modified nucleotides can be aminoallyl (aa)-dUTP,aa-dCTP, aa-dGTP, and/or aa-dATP.

In some embodiments, after generation of the extended capture probe, thesecond strand, or both the extended capture probe and/or the secondstrand can be released from the surface of the substrate. For example,the extended capture probe and/or the second strand can be released byany of the methods described herein (e.g., heat or cleavage via acleavage domain). In some embodiments, the amine-modified nucleotidesincorporated into the extended capture probe can be cross-linked to thesurface of a substrate or cross-linked to the biological sample usingits amine-modified nucleotides. In some embodiments, the surface of thesubstrate can be coated in a hydrogel. In some embodiments, the surfaceof the substrate can be coated in a protein matrix. In some embodiments,the cross-linking can be irreversible. In some embodiments, thecross-linked extended capture probe and/or second strand can becircularized. For example, circular template ligation can be performedby a DNA ligase (e.g., T4 DNA ligase) or circular template-free ligationcan be performed by a template independent ligase (e.g., CircLigase). Insome embodiments, the extended capture probe is circularized withCircLigase. In some embodiments, the circularized extended capture probecan be amplified. For example, rolling circle amplification can beperformed with a suitable DNA polymerase (e.g., phi29). In someembodiments, the capture probe includes a functional domain (e.g.,sequencing adapter). In some embodiments, rolling circle amplificationcan be performed with a primer complementary to the functional domain(e.g., sequencing adapter). In some embodiments, the rolling circleamplification can be performed to generate two or more amplicons (e.g.,one or more amplicons including any of the amine-modified nucleotidesdescribed herein). In some embodiments, the two or more ampliconsproduced by the rolling circle amplification can be cross-linked to thesurface of the substrate and/or cross-linked to the biological sample.In some embodiments, the two or more amplicons can be sequenced in situ.The in situ sequencing can be performed by any method described herein(See, Lee, J. H., Fluorescent in situ sequencing (FISSEQ) of RNA forgene expression profiling, Nat Protoc., 10(3): 442-458,doi:10.1038/nprot.2014.191 (2015), which is incorporated herein byreference). In some embodiments, the two or more amplicons can beimaged.

In some embodiments, spatial analysis by any of the methods describedherein can be performed on ribosomal RNA (rRNA), including, endogenousribosomal RNA (e.g., native to the biological sample), and/or exogenousRNA (e.g., microbial ribosomal RNA and/or viral RNA also present in thebiological sample). As used herein, “metagenomics,” can refer to thestudy of exogenous nucleic acids (e.g., DNA, RNA, or other nucleic acidsdescribed herein) present in a biological sample. As used herein,“spatial metagenomics,” can refer to the study of the spatial locationof exogenous nucleic acid present in a biological sample. Spatialmetagenomics can also refer to the identification of one or more species(e.g., viral or microbial) present in the biological sample and/or thestudy of identifying patterns of proximity (e.g., co-localization)amongst species.

In some embodiments, microbial rRNA can be spatially detected,quantified, and/or amplified from a biological sample. In someembodiments, rRNA (e.g., 16S ribosomal RNA) can be associated with aparticular microbial species. For example, microbial ribosomal RNA(e.g., 16S ribosomal RNA) can be used to identify one or more species ofmicrobe present in the biological sample (See e.g., Kolbert, C. P., andPersing, D. H., Ribosomal DNA sequencing as a tool for identification ofbacterial pathogens, Current Opinion in Microbiology. 2 (3): 299-305.doi:10.1016/S1369-5274(99)80052-6. PMID 10383862 (1999), which isincorporated herein by reference). In some embodiments, identificationof microbial species in proximity to one or more other microbial speciescan be identified.

In some embodiments, a biological sample be covered (e.g., coated) orembedded in with a photo-crosslinkable coating (e.g., conditionallydissolvable polymer, e.g., DTT sensitive hydrogel). A biological samplecan be contacted with the photo-crosslinkable coated substrate. In someembodiments, the biological sample and photo-crosslinkable substrate areassembled into a flow-cell and the photo-crosslinkable polymer can beincubated with the biological sample. The biological sample can becross-linked into hydrogel-voxels of defined dimensions using a lightsource and a photomask. In some embodiments, the flow-cell can bedismantled and washed to remove unpolymerized hydrogel. Thephoto-crosslinkable coating can be treated with DTT to yield single-cellportions or approximately single-cell portions.

In some embodiments, the single-cell or approximately single-cellportions can be encapsulated in a vesicle. The vesicle can contain abarcoded feature (e.g., a bead), and the barcoded feature can contain acapture domain. In some embodiments, the capture domain can bindspecifically to microbial rRNA (e.g., microbial 16S rRNA). In someembodiments, the captured microbial rRNA can be amplified and analyzed(e.g., sequenced) by any of the methods described herein. In someembodiments, the amplified and sequenced microbial rRNA can identifymicrobial species and/or patterns of proximity (e.g., co-localization)of one or more species.

Alternatively, spatial analysis can be performed on exogenous rRNA(e.g., microbial or viral) with a plurality of capture probes on asubstrate (e.g., an array), wherein the capture probes include a spatialbarcode and a capture domain. In some embodiments, the capture domaincan be configured to interact (e.g., hybridize) with microbial rRNApresent in the biological sample. The capture probe can be configured tointeract with any microbial rRNA. In some embodiments, the capture probeis configured to interact with microbial 16S rRNA. The biological samplecan be treated (e.g., permeabilized) such that the capture domain andthe analyte (e.g., microbial rRNA) interact (e.g., hybridize). In someembodiments, the captured analyte (e.g., microbial rRNA) can be reversetranscribed generating an extended capture probe, followed generation ofa second strand that is complementary to the extended capture probe asdescribed herein. The extended capture probe and/or the second strandcan include a portion or all of a capture probe sequence, or acomplement thereof. The capture probe sequence, or complement thereof,can include the spatial barcode, or complement thereof. In someembodiments, the first strand cDNA, and optionally, the second strandcDNA can be amplified by any method described herein. The amplifiedcapture probes and analytes can be analyzed (e.g., sequenced) by anymethod described herein. The spatial information of thespatially-barcoded features can be used to determine the spatiallocation of the captured analytes (e.g., microbial rRNA) in thebiological sample, or a portion thereof. In some embodiments, thecaptured analyte can identify the microbial species present in thebiological sample, or a portion thereof. In some embodiments, thespatial information and identity of microbial species present in thebiological sample can be correlated with one another, thus revealingwhether certain microbial species may be found in proximity (e.g.,co-localize) with one another in the biological sample.

In exemplary embodiments, provided herein are methods for detecting anucleic acid within a portion of biological sample that include: (a)immobilizing the biological sample in a gel matrix to produce anembedded biological sample; (b) breaking up the embedded biologicalsample into portions; (c) lysing cell(s) present in the portions; (d)encapsulating a portion from step (c) together with a bead having anattached capture probe comprising a spatial barcode and a capture domainthat binds specifically to the nucleic acid in the portion; and (e)determining (i) all or a part of the sequence of the spatial barcode, ora complement thereof, and (ii) all or part of the sequence of thenucleic acid, or a complement thereof, and using the determinedsequences of (i) and (ii) to detect the nucleic acid within the portionof the biological sample. In some embodiments of these methods, thenucleic acid comprises microbial ribosomal RNA (rRNA). In someembodiments of these methods, the microbial rRNA comprises 16S rRNA. Insome embodiments of these methods, the method comprises detecting 16SrRNA from at least two different microbacteria within the portion of thebiological sample. In some embodiments of these methods, the nucleicacid is an mRNA.

Provided herein are methods for spatially profiling analytes within abiological sample. Profiles of biological samples (e.g., individualcells, populations of cells, tissue sections, etc.) can be compared toprofiles of other cells, e.g., “normal,” or “healthy,” biologicalsamples. In some embodiments of any the methods for spatially profilinganalytes described herein, the method can provide for diagnosis of adisease (e.g., cancer, Alzheimer's disease, Parkinson's disease). Insome embodiments of any the methods for spatially profiling analytesdescribed herein, the methods can be used in drug screening. In someembodiments of any the methods for spatially profiling analytesdescribed herein, the methods can be used to perform drug screening withan organoid. In some embodiments of any the methods for spatiallyprofiling analytes described herein, the methods can be used to detectchanges in (e.g., altered) cellular signaling. In some embodiments ofany the methods for spatially profiling analytes described herein, themethods can include the introduction of a pathogen to the biologicalsample and evaluation of the response of the biological sample to thepathogen. In some embodiments of any the methods for spatially profilinganalytes described herein, the methods include exposing the biologicalsample to a perturbation agent (e.g., any of the perturbation agentsdescribed herein) and evaluating the response of the biological sampleto the perturbation agent. In some embodiments of any the methods forspatially profiling analytes described herein, the methods includemonitoring cell differentiation in a biological sample (e.g., anorganoid). In some embodiments of any the methods for spatiallyprofiling analytes described herein, the methods include analyzingtissue morphogenesis. In some embodiments of any the methods forspatially profiling analytes described herein, the methods includeidentifying spatial heterogeneity in a biological sample (e.g.,identifying different cell types or populations in a biological sample).In some embodiments of any the methods for spatially profiling analytesdescribed herein, the methods include analyzing the spatiotemporal order(e.g., timing) of molecular events. For example, the methods forspatially profiling analytes can include monitoring expression levelsover the course of a disease.

The methods provided herein can also be used to determine a relativelevel of inflammation in a subject (e.g., determine an inflammatoryscore) or a subject's response to treatment or the development ofresistance to treatment. The methods described herein can also be usedto identify candidate targets for potential therapeutic interventionand/or to identify biomarkers associated with different disease statesin a subject.

(h) Quality Control

(i) Control Sample

As used herein, the term “control sample” typically refers to asubstrate that is insoluble in aqueous liquid and that allows for anaccurate and traceable positioning of test analytes on the substrate.The term “control sample” and “test substrate” are used interchangeablyherein. The control sample can be any suitable substrate known to theperson skilled in the art. Exemplary control samples comprise asemi-porous material. Non-limiting examples of a semi-porous materialinclude a nitrocellulose membrane, a hydrogel, and a nylon filter.

A control sample or test substrate can be of any appropriate dimensionor volume (e.g., size or shape). In some embodiments, a control sampleis a regular shape (e.g., a square, circle, or a rectangle). In someembodiments, a surface of a control sample has any appropriate form orformat. For example, the surface of a control sample can be flat orcurved (e.g., convexly or concavely curved towards the area where theinteraction between the substrate and the control sample takes place).In some embodiments, a control sample has rounded corners (e.g., forincreased safety or robustness). In some embodiments, a control samplehas one or more cut-off corners (e.g., for use with a slide clamp orcross-table).

A control sample can comprise a plurality of test analytes. In someembodiments, the members of the plurality of test analytes are disposedon the substrate in a known amount and in a known location. For example,a plurality of test analytes are disposed at a known amount on thecontrol sample at one or more locations. In some embodiments, theplurality of test analytes are disposed on the substrate in a definedpattern (e.g., an x-y grid pattern). In some embodiments, the definedpattern includes one or more locations or spots.

In some embodiments, each location comprises a plurality of the samespecies of test analyte. In some embodiments, each location comprises aplurality of one or more different species of test analytes. In someembodiments, each location on the control sample represents a differentregion of a biological sample, e.g., a tissue sample. In someembodiments, an area on the control sample that does not comprise aplurality of test analytes represents an area where no biological sampleis present.

In some embodiments, the plurality of test analytes comprises one ormore test analytes,

e.g., a first test analyte, a second test analyte, a third test analyte,a fourth test analyte, etc. In some embodiments, the plurality of testanalytes comprises nucleic acids. In some embodiments, each location orfeature comprises a population of nucleic acid sequences. In someembodiments, the nucleic acid sequence of a first test analyte differsfrom the nucleic acid sequence of a second test analyte by a singlenucleic acid residue. In some embodiments, each location or featurecomprises a population of RNA transcripts and one or more specificsurface marker proteins or one or more CRISPR guide RNAs. In someembodiments, the plurality of test analytes comprises a bacterialartificial chromosomes (BAC). In some embodiments, each location on thecontrol sample comprises a unique blend of BACs. In some embodiments,proteins are cross-linked to the BACs, for example, to mimic histonebinding on DNA.

In some embodiments, the concentration of a first test analyte differsfrom the concentration of a second test analyte at a different locationor feature on the control sample. In some embodiments, the first testanalyte and the second test analyte comprise an identical nucleotidesequence.

A control sample can be used to determine process bias. Barcoded arrayscan be placed on top of a control sample comprising a plurality of testanalytes, where members of the plurality of test analytes can bedisposed on a substrate in a known amount and in a known location. Thearray can then be removed, and the molecular information therein can bedetermined by performing library construction for next generationsequencing, followed by computational analysis to correlate theexpression values of the test analytes with the barcodes (e.g., spatialbarcodes) on the array. The sequencing data can be compared with theknown amount and the known locations of the plurality of test analytesto determine whether the spatial analysis workflow accurately detectsthe presence, amount, location, or combinations thereof, of the testanalyte, thereby determining process bias of the spatial analysisworkflow.

(ii) RNA Integrity Number (RIN)

As used herein, the term “RNA Integrity Number” or “RIN” refers to thein situ indication of RNA quality based on an integrity score. HigherRIN scores correlate with higher data quality in the spatial profilingassays described herein. For example, a first biological sample with ahigh RIN score will have higher data quality compared to a secondbiological sample with RIN score lower than the first biological sample.In some embodiments, a RIN is calculated for a tissue section, one ormore regions of a tissue section, or a single cell.

In some embodiments, one or more RINs for a given biological sample(e.g., tissue section, one or more regions of a tissue, or a singlecell) are calculated by: (a) providing (i) a spatial array including aplurality of capture probes on a substrate, where a capture probecomprises a capture domain and (ii) a tissue stained with a histologystain (e.g., any of the stains described herein); (b) contacting thespatial array with the biological sample (e.g., tissue); (c) capturing abiological analyte (e.g., an 18S rRNA molecule) from the biologicalsample (e.g., tissue) with the capture domain; (d) generating a cDNAmolecule from the captured biological analyte (e.g., 18S rRNA); (e)hybridizing one or more labeled oligonucleotide probes to the cDNA; (f)imaging the labeled cDNA and the histology stain (e.g., any of thestains described herein), and (g) generating a RNA integrity number fora location in the spatial array, wherein the RNA integrity numbercomprises an analysis of a labeled cDNA image and a histology stain(e.g., any of the stains described herein) image for the location.

In some embodiments, the biological sample (e.g., tissue) is stainedwith a histology stain. As used herein, a “histology stain” can be anystain described herein. For example, the biological sample can bestained with IF/IHC stains described herein. For example, the biologicalsample (e.g., tissue) can be stained with hematoxylin and eosin (“H&E”).In some embodiments, the biological sample (e.g., tissue) is stainedwith a histology stain (e.g., any of the stains described herein)before, contemporaneously with, or after labelling of the cDNA withlabeled oligonucleotide probes. In some embodiments, the stainedbiological sample can be, optionally, destained (e.g., washed 1, 2, 3,4, 5, or more times in a low pH acid (e.g., HCl)). For example,hematoxylin, from an H&E stain, can be optionally removed from thebiological sample by washing in dilute HCl (0.001M to 0.1M) prior tofurther processing. In some embodiments, the stained biological samplecan be optionally destained after imaging and prior to permeabilization.

In some embodiments, the spatial array includes a plurality of captureprobes immobilized on a substrate where the capture probes include atleast a capture domain. In some embodiments, the capture domain includesa poly(T) sequence. For example, a capture domain includes a poly(T)sequence that is capable of capturing an 18S rRNA transcript from abiological sample.

In some embodiments, calculating one or more RNA Integrity Numbers for abiological sample includes hybridizing at least one (e.g., at least two,at least three, at least four, or at least five) labeled oligonucleotideprobes to the cDNA generated from the 18s rRNA. In some embodiments, alabeled oligonucleotide probe includes a sequence that is complementaryto a portion of the 18S cDNA. In some embodiments, four labeledoligonucleotide probes (P1-P4) are designed to hybridize at fourdifferent locations spanning the entire gene body of the 18S rRNA. Insome embodiments, a labeled oligonucleotide probe can include any of thedetectable labels as described herein. For example, an oligonucleotidelabeled probe can include a fluorescent label (e.g., Cy3). In someembodiments, one or more of the labeled oligonucleotide probes designedwith complementarity to different locations within the 18S cDNA sequenceinclude the same detectable label. For example, four labeledoligonucleotide probes, (P1-P4) each designed to have complementarity toa different location within the 18S cDNA sequence can all have the samedetectable label (e.g., Cy3). In some embodiments, one or more of thelabeled oligonucleotide probes designed with complementarity todifferent locations within the 18S cDNA sequence include a differentdetectable label. For example, four labeled oligonucleotide probes,(P1-P4) each designed to have complementarity to a different locationwithin the 18S cDNA sequence can include different detectable labels.

In some embodiments, determining a RNA Integrity Number for a biologicalsample (e.g., tissue section, one or more regions of a tissue, or asingle cell) includes analyzing the images taken from a spatial arrayand a histology stain (e.g., any of the stains described herein) for thesame location. For example, for the spatial array, all images aregenerated by scanning with a laser (e.g., a 532 nm wavelength) after thefluorescently labeled (e.g., Cy3) oligonucleotide probes have beenhybridized to the 18S cDNA. One image is generated per probe (P1-P4) andone image is generated where no fluorescently labeled probes werehybridized (P0). Normalization of Fluorescence Units (FU) data isperformed by subtraction of the auto-fluorescence recorded with PO anddivision with P1. After alignment, the five images (one image from eachprobe, P1-P4, and one image from an area without bound probe) are loadedinto a script. The script generates two different plots, one heat-map ofRIN values and one image alignment error plot, which combines thehistology stain (e.g., any of the stains described herein) image. Theimage alignment error plot is used to visualize which pixels andpositions should be excluded from the analysis due to alignment errorsbetween the images from P0-P4.

III. General Spatial Cell-Based Analytical Methodology

(a) Barcoding a Biological Sample

In some embodiments, provided herein are methods and materials forattaching and/or introducing a molecule (e.g., a peptide, a lipid, or anucleic acid molecule) having a barcode (e.g., a spatial barcode) to abiological sample (e.g., to a cell in a biological sample) for use inspatial analysis. In some embodiments, a plurality of molecules (e.g., aplurality of nucleic acid molecules) having a plurality of barcodes(e.g., a plurality of spatial barcodes) are introduced to a biologicalsample (e.g., to a plurality of cells in a biological sample) for use inspatial analysis.

FIG. 18 is a schematic diagram depicting cell tagging using eithercovalent conjugation of an analyte binding moiety to a cell surface ornon-covalent interactions with cell membrane elements. FIG. 18 listsnon-exhaustive examples of a covalent analyte binding moiety/cellsurface interactions, including protein targeting, amine conjugationusing NHS chemistry, cyanuric chloride, thiol conjugation via maleimideaddition, as well as targeting glycoproteins/glycolipids expressed onthe cell surface via click chemistry. Non-exhaustive examples ofnon-covalent interactions with cell membrane elements include lipidmodified oligos, biocompatible anchor for cell membrane (BAM, e.g.,oleyl-PEG-NHS), lipid modified positive neutral polymer, and antibody tomembrane proteins. A cell tag can be used in combination with an analytecapture agent and cleavable or non-cleavable spatially-barcoded captureprobes for spatial and multiplexing applications.

In some embodiments, a plurality of molecules (e.g., a plurality oflipid or nucleic acid molecules) having a plurality of barcodes (e.g., aplurality of spatial barcodes) are introduced to a biological sample(e.g., to a plurality of cells in a biological sample) for use inspatial analysis, wherein the plurality of molecules are introduced tothe biological sample in an arrayed format. In some embodiments, aplurality of molecules (e.g., a plurality of lipid or nucleic acidmolecules) having a plurality of barcodes are provided on a substrate(e.g., any of the variety of substrates described herein) in any of thevariety of arrayed formats described herein, and the biological sampleis contacted with the molecules on the substrate such that the moleculesare introduced to the biological sample. In some embodiments, themolecules that are introduced to the biological sample are cleavablyattached to the substrate, and are cleaved from the substrate andreleased to the biological sample when contacted with the biologicalsample. In some embodiments, the molecules introduced to the biologicalsample are covalently attached to the substrate prior to cleavage. Insome embodiments, the molecules that are introduced to the biologicalsample are non-covalently attached to the substrate (e.g., viahybridization), and are released from the substrate to the biologicalsample when contacted with the biological sample.

In some embodiments, a plurality of molecules (e.g., a plurality oflipid or nucleic acid molecules) having a plurality of barcodes (e.g., aplurality of spatial barcodes) are migrated or transferred from asubstrate to cells of a biological sample. In some embodiments,migrating a plurality of molecules from a substrate to cells of abiological sample includes applying a force (e.g., mechanical,centrifugal, or electrophoretic) to the substrate and/or the biologicalsample to facilitate migration of the plurality of molecules from thesubstrate to the biological sample.

In some embodiments of any of the spatial analysis methods describedherein, physical force is used to facilitate attachment to orintroduction of a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) into a biological sample (e.g., a cellpresent in a biological sample). As used herein, “physical force” refersto the use of a physical force to counteract the cell membrane barrierin facilitating intracellular delivery of molecules. Examples ofphysical force instruments and methods that can be used in accordancewith materials and methods described herein include the use of a needle,ballistic DNA, electroporation, sonoporation, photoporation,magnetofection, hydroporation, and combinations thereof.

(i) Introducing a Cell-Tagging Agent to the Surface of a Cell

In some embodiments, biological samples (e.g., cells in a biologicalsample) can be labelled using cell-tagging agents where the cell-taggingagents facilitate the introduction of the molecules (e.g., nucleic acidmolecules) having barcodes (e.g., spatial barcodes) into the biologicalsample (e.g., into cells in a biological sample). As used herein, theterm “cell-tagging agent” refers to a molecule having a moiety that iscapable of attaching to the surface of a cell (e.g., thus attaching thebarcode to the surface of the cell) and/or penetrating and passingthrough the cell membrane (e.g., thus introducing the barcode to theinterior of the cell). In some embodiments, a cell-tagging agentincludes a barcode (e.g., a spatial barcode). The barcode of a barcodedcell-tagging agent can be any of the variety of barcodes describedherein. In some embodiments, the barcode of a barcoded cell-taggingagent is a spatial barcode. In some embodiments, a cell-tagging agentcomprises a nucleic acid molecule that includes the barcode (e.g., thespatial barcode). In some embodiments, the barcode of a barcodedcell-tagging agent identifies the associated molecule, where eachbarcode is associated with a particular molecule. In some embodiments,one or more molecules are applied to a sample. In some embodiments, anucleic acid molecule that includes the barcode is covalently attachedto the cell-tagging agent. In some embodiments, a nucleic acid moleculethat includes the barcode is non-covalently attached to the cell-taggingagent. A non-limiting example of non-covalent attachment includeshybridizing the nucleic acid molecule that includes the barcode to anucleic acid molecule on the cell-tagging agent (which nucleic acidmolecule on the cell-tagging agent can be bound to the cell-taggingagent covalently or non-covalently). In some embodiments, a nucleic acidmolecule attached to a cell-tagging agent that includes a barcode (e.g.,a spatial barcode) also includes one or more additional domains. Suchadditional domains include, without limitation, a PCR handle, asequencing priming site, a domain for hybridizing to another nucleicacid molecule, and combinations thereof.

In some embodiments, a cell-tagging agent attaches to the surface of acell. When the cell-tagging agent includes a barcode (e.g., a nucleicacid that includes a spatial barcode), the barcode is also attached tothe surface of the cell. In some embodiments of any of the spatialanalysis methods described herein, a cell-tagging agent attachescovalently to the cell surface to facilitate introduction of the spatialanalysis reagents. In some embodiments of any of the spatial analysismethods described herein, a cell-tagging agent attaches non-covalentlyto the cell surface to facilitate introduction of the spatial analysisreagents.

In some embodiments, once a cell or cells in a biological sample isspatially tagged with a cell-tagging agent(s), spatial analysis ofanalytes present in the biological sample is performed. In someembodiments, such spatial analysis includes dissociating thespatially-tagged cells of the biological sample (or a subset of thespatially-tagged cells of the biological sample) and analyzing analytespresent in those cells on a cell-by-cell basis. Any of a variety ofmethods for analyzing analytes present in cells on a cell-by-cell basiscan be used. Non-limiting examples include any of the variety of methodsdescribed herein and methods described in PCT Application PublicationNo. WO 2019/113533A1, the content of which is incorporated herein byreference in its entirety. For example, the spatially-tagged cells canbe encapsulated with beads comprising one or more nucleic acid moleculeshaving a barcode (e.g., a cellular barcode) (e.g., an emulsion). Thenucleic acid present on the bead can have a domain that hybridizes to adomain on a nucleic acid present on the tagged cell (e.g., a domain on anucleic acid that is attached to a cell-tagging agent), thus linking thespatial barcode of the cell to the cellular barcode of the bead. Oncethe spatial barcode of the cell and the cellular barcode of the bead arelinked, analytes present in the cell can be analyzed using captureprobes (e.g., capture probes present on the bead). This allows thenucleic acids produced (using these methods) from specific cells to beamplified and sequenced separately (e.g., within separate partitions ordroplets).

In some embodiments, once a cell or cells in a biological sample isspatially tagged with a cell-tagging agent(s), spatial analysis ofanalytes present in the biological sample is performed in which thecells of the biological sample are not dissociated into single cells. Insuch embodiments, various methods of spatial analysis such as any ofthose provided herein can be employed. For example, once a cell or cellsin a biological sample is spatially tagged with a cell-tagging agent(s),analytes in the cells can be captured and assayed. In some embodiments,cell-tagging agents include both a spatial barcode and a capture domainthat can be used to capture analytes present in a cell. For example,cell-tagging agents that include both a spatial barcode and a capturedomain can be introduced to cells of the biological sample in a way suchthat locations of the cell-tagging agents are known (or can bedetermined after introducing them to the cells). One non-limitingexample of introducing cell-tagging agents to a biological sample is toprovide the cell-tagging agents in an arrayed format (e.g., arrayed on asubstrate such as any of the variety of substrates and arrays providedherein), where the positions of the cell-tagging agents on the array areknown at the time of introduction (or can be determined afterintroduction). The cells can be permeabilized as necessary (e.g., usingpermeabilization agents and methods described herein), reagents foranalyte analysis can be provided to the cells (e.g., a reversetranscriptase, a polymerase, nucleotides, etc., in the case where theanalyte is a nucleic acid that binds to the capture probe), and theanalytes can be assayed. In some embodiments, the assayed analytes(and/or copies thereof) can be released from the substrate and analyzed.In some embodiments, the assayed analytes (and/or copies thereof) areassayed in situ.

Non-limiting examples of cell-tagging agents and systems that attach tothe surface of a cell (e.g., thus introducing the cell-tagging agent andany barcode attached thereto to the exterior of the cell) that can beused in accordance with materials and methods provided herein forspatially analyzing an analyte or analytes in a biological sampleinclude: lipid tagged primers/lipophilic-tagged moieties, positive orneutral oligo-conjugated polymers, antibody-tagged primers,streptavidin-conjugated oligonucleotides, dye-tagged oligonucleotides,click-chemistry, receptor-ligand systems, covalent binding systems viaamine or thiol functionalities, and combinations thereof

-   -   (ii) Introducing a Cell-Tagging Agent to the Interior of a Cell

Non-limiting examples of cell-tagging agents and systems that penetrateand/or pass through the cell membrane (e.g., thus introducing thecell-tagging agent and any barcode attached thereto to the interior ofthe cell) that can be used in accordance with materials and methodsprovided herein for spatially profiling an analyte or analytes in abiological sample include: a cell-penetrating agent (e.g., acell-penetrating peptide), a nanoparticle, a liposome, a polymersome, apeptide-based chemical vector, electroporation, sonoporation, lentiviralvectors, retroviral vectors, and combinations thereof.

In some embodiments, a cell-tagging agent comprises a cell-penetratingagent (described below). In some embodiments, a cell-penetrating agenttransports the cell-tagging agent into the cells of a biological sample.When a cell-tagging agent comprises a barcode (e.g., a nucleic acid thatincludes a spatial barcode), the barcode also penetrates into the cell.In some embodiments, a plurality of cell-tagging agents are cleaved(e.g., photocleaved) from an array via a cleavage domain, thus freeingthe cell-tagging agents from the array and allowing at least one captureprobe of the plurality to penetrate a cell. The cell-tagging agent canthen interact with an intracellular biological analyte via the capturedomain. In some embodiments, the plurality of capture probes is migratedfrom the array into cells of the biological sample via cell-penetratingagents. In some embodiments, migrating a plurality of capture probesfrom the array to cells of the biological sample includes applying aforce (e.g., mechanical, centrifugal, or electrophorectic) to thebiological sample.

In some embodiments, the biological sample is treated with one or morereagents to facilitate migration of a plurality of freed (e.g., cleaved)capture probes into cells of a biological sample. In one embodiment, anorganic solvent (e.g., methanol or acetone) may be used to permeabilizecells of a biological sample. In another embodiment, a detergent (e.g.,saponon, Triton X-100™, or Tween-20™) may be used to permeabilize cellsof a biological sample. In yet another embodiment, an enzyme (e.g.,trypsin) may be used to permeabilize cells of a biological sample. Anysuitable method of cell permeabilization may be used to practice themethods disclosed herein. In some embodiments, the biological sample canbe incubated with a cellular permeabilization reagent after contactingthe array with the biological sample. In some embodiments, thebiological sample can be fixed according to methods described herein.

In some embodiments, migrating a plurality of freed (e.g., cleaved)capture probes into cells of a biological sample includes passivemigration (e.g., diffusion). In some embodiments, migrating a pluralityof freed (e.g. cleaved) capture probes into cells of a biological sampleincludes active migration (e.g., electrophoretic migration). In someembodiments, migrating a plurality of freed (e.g., cleaved) captureprobes into cells of a biological sample comprises antibodies. In someembodiments, migrating a plurality of freed (e.g., cleaved) captureprobes into cells of a biological sample comprises transfection (e.g.,chemical, biological, physical, viral vectors).

FIG. 19 is a schematic showing an exemplary cell tagging method.Non-exhaustive examples of oligo delivery vehicles may include a cellpenetrating peptide or a nanoparticle. Non-exhaustive examples of thedelivery systems can include lipid-based polymeric and metallicnanoparticles or oligos that can be conjugated or encapsulated withinthe delivery system. The cell tag can be used in combination with acapture agent barcode domain and a cleavable or non-cleavablespatially-barcoded capture probes for spatial and multiplexingapplications.

1. Cell-Penetrating Agent

In some embodiments of any of the spatial profiling methods describedherein, identification of a biological analyte by a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain is facilitated by a cell-penetrating agent. In someembodiments, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) and a capture domain is coupled to acell-penetrating agent, and the cell-penetrating agent allows themolecule to interact with an analyte inside the cell. A“cell-penetrating agent” as used herein can refer to an agent capable offacilitating the introduction of a molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and a capturedomain into a cell of a biological sample (see, e.g., Lovatt et al. NatMethods. 2014 Feb.; 11(2):190-6, which is incorporated herein byreference in its entirety). In some embodiments, a cell-penetratingagent is a cell-penetrating peptide. A “cell-penetrating peptide” asused herein refers to a peptide (e.g., a short peptide, e.g., a peptidenot usually exceeding 30 residues) that has the capacity to crosscellular membranes. In some embodiments, cell-penetrating agents or cellpenetrating peptides may be covalently or non-covalently coupled to amolecule (e.g., a barcoded nucleic acid molecule), likely at the 5′ endof the molecule. A cell-penetrating peptide may direct the barcodednucleic acid molecule to a specific organelle.

In some embodiments of any of the spatial profiling methods describedherein, a cell-penetrating peptide coupled to a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain can cross a cellular membrane using an energy dependentor an energy independent mechanism. For example, a cell-penetratingpeptide can cross a cellular membrane through direct translocationthrough physical perturbation of the plasma membrane, endocytosis (e.g.,mediated via clathrin), adaptive translocation, pore-formation,electroporation-like permeabilization, and/or entry at microdomainboundaries. Non-limiting examples of a cell-penetrating peptide include:penetratin, tat peptide, pVEC, transportan, MPG, Pep-1, a polyargininepeptide, MAP, R6W3, (D-Arg)9, Cys(Npys)-(D-Arg)9, Anti-BetaGamma(MPS-Phosducin-like protein C terminus), Cys(Npys) antennapedia,Cys(Npys)-(Arg)9, Cys(Npys)-TAT (47-57), HIV-1 Tat (48-60), KALA,mastoparan, penetratin-Arg, pep-1-cysteamine, TAT(47-57)GGG-Cys(Npys),Tat-NR2Bct, transdermal peptide, SynBl, SynB3, PTD-4, PTD-5, FHVCoat-(35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), R9-tat, SBP, FBP, MPG,MPG(ΔNLS), Pep-2, MTS, pls1, and a polylysine peptide (see, e.g.,Bechara et al. FEBS Lett. 2013 Jun. 19; 587(12):1693-702, which isincorporated by reference herein in its entirety).

In some embodiments, there could be two orientations forcell-penetrating peptide (CPP) conjugation. For example, one orientationcan be (N-terminus)-CPP-Cys-(C-terminus)-linker-NH2C6-5′-oligo-3′;3′-oligo-5′-NH2C6-linker-(N-terminus)-Cys-CPP-(C-terminus). The methodsherein can be performed with other CPP conjugations and orientations.

In some embodiments, cell-tagging agents further comprise acell-penetration tag. A “cell-penetration tag” as used herein referes toan agent that can be detected as inside a cell. In some embodiments, acell penetration tag includes a fluorophore. In some embodiments, a cellpenetration tag is selected from the group consisting of: Cy2, Cy3,Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein (6-FAM), DyLight, AlexaFluor®, and tetramethylrhodamine (TAMRA) azide.

In some embodiments, a cell penetration tag is detected. In someembodiments, a cell penetration tag is detected after releasing aplurality of capture probes from the array and removing the array fromthe biological sample. In some embodiments, introduction of acell-tagging agent into the cell is determined by detecting the presenceof the cell penetration tag in the cell.

In some embodiments, cell-tagging agents can optionally include anintracellular cleavage domain, wherein one or more segments or regionsof the capture probe (e.g., capture domains, spatial barcodes, and/orUMIs) can be releasably or cleavably bound to the cell-penetratingagent, such that the capture domain, spatial barcode, and/or UMI can bereleased. In some embodiments, the cleavage of the linkage between thecapture domain, spatial barcode, and/or UMI and the cell-penetratingagent is induced in an intracellular environment (e.g., theintracellular cleavage domain is cleaved after the cell-tagging agent isintroduced into the cell). In some embodiments, the intracellularcleavage domain comprises a disulfide bond. For example, theintracellular cleavage domain can be a disulfide bond cleaved byreducing conditions in the cell. Any other suitable linker can be usedto release or cleave the intracellular cleavage domain of the captureprobe.

2. Nanoparticles

In some embodiments of any of the spatial profiling methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by an inorganic particle (e.g., a nanoparticle).In some embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) and a capture domain is coupled to aninorganic particle (e.g., a nanoparticle), and the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain uses the nanoparticle to get access to analytes insidethe cell. Non-limiting examples of nanoparticles that can be used inembodiments herein to deliver a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) and a capture domain into acell and/or cell bead include inorganic nanoparticles prepared frommetals, (e.g., iron, gold, and silver), inorganic salts, and ceramics(e.g., phosphate or carbonate salts of calcium, magnesium, or silicon).The surface of a nanoparticle can be coated to facilitate binding of themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain, or the surface can be chemicallymodified to facilitate attachment of the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and a capturedomain. Magnetic nanoparticles (e.g., supermagnetic iron oxide),fullerenes (e.g., soluble carbon molecules), carbon nanotubes (e.g.,cylindrical fullerenes), quantum dots, and supramolecular systems canalso be used.

3. Liposomes

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a liposome. Various types of lipids, includingcationic lipids, can be used in liposome delivery. In some cases, amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain is delivered to a cell via a lipidnano-emulsion. A lipid emulsion refers to a dispersion of one immiscibleliquid in another stabilized by emulsifying agent. Labeling cells cancomprise use of a solid lipid nanoparticle.

4. Polymersomes

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a polymersome. In some embodiments, a molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) and a capture domain is contained in the polymersome, and themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain uses the polymersome to get accessto analytes inside the cell. A “polymersome” as referred to herein is anartificial vesicle. For example, a polymersome can be a vesicle similarto a liposome, but the membrane comprises amphiphilic synthetic blockcopolymers (see, e.g., Rideau et al. Chem. Soc. Rev., 2018, 47,8572-8610, which is incorporated by reference herein in its entirety).In some embodiments, polymersomes comprise di-(AB) or tri-blockcopolymers (e.g., ABA or ABC), where A and C are a hydrophilic block andB is a hydrophobic block. In some embodiments, a polymersome comprisespoly(butadiene)-b-poly(ethylene oxide), poly(ethylethylene)-b-poly(ethylene oxide), polystyrene-b-poly(ethylene oxide),poly(2-vinylpyridine)-b-poly(ethylene oxide),polydimethylsiloxane-b-poly(ethylene oxide),polydimethylsiloxane-g-poly(ethylene oxide),polycaprolactone-b-poly(ethylene oxide), polyisobutylene-b-poly(ethyleneoxide), polystyrene-b-polyacrylic acid,polydimethylsiloxane-b-poly-2-methyl-2-oxazoline, or a combinationthereof (wherein b=block and g=grafted).

5. Peptide-Based Chemical Vectors

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a peptide-based chemical vector, e.g., acationic peptide-based chemical vector. Cationic peptides can be rich inbasic residues like lysine and/or arginine. In some embodiments of anyof the spatial analysis methods described herein, capture of abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain is facilitatedby a polymer-based chemical vector. Cationic polymers, when mixed with amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain, can form nanosized complexescalled polyplexes. Polymer based vectors can comprise natural proteins,peptides and/or polysaccharides. Polymer based vectors can comprisesynthetic polymers. In some embodiments, a polymer-based vectorcomprises polyethylenimine (PEI). PEI can condense DNA intopositively-charged particles, which bind to anionic cell surfaceresidues and are brought into the cell via endocytosis. In someembodiments, a polymer-based chemical vector comprises poly(L)-lysine(PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside)(PLGA), polyornithine, polyarginine, histones, protamines, or acombination thereof. Polymer-based vectors can comprise a mixture ofpolymers, for example, PEG and PLL. Other non-limiting examples ofpolymers include dendrimers, chitosans, synthetic amino derivatives ofdextran, and cationic acrylic polymers.

6. Electroporation

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by electroporation. With electroporation, abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain can enter acell through one or more pores in the cellular membrane formed byapplied electricity. The pore of the membrane can be reversible based onthe applied field strength and pulse duration.

7. Sonoporation

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by sonoporation. Cell membranes can be temporarilypermeabilized using sound waves, allowing cellular uptake of abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain.

8. Lentiviral Vectors and Retroviral Vectors

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by vectors. For example, a vector as describedherein can be an expression vector where the expression vector includesa promoter sequence operably linked to the sequence encoding themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain. Non-limiting examples of vectorsinclude plasmids, transposons, cosmids, and viral derived vectors (e.g.,any adenoviral derived vectors (AV) cytomegaloviral derived (CMV)vectors, simian viral derived (SV40) vectors, adeno-associated virus(AAV) vectors, lentivirus vectors, and retroviral vectors), and anyGateway® vectors. A vector can, for example, include sufficientcis-acting elements for expression where other elements for expressioncan be supplied by the host mammalian cell or in an in vitro expressionsystem. Skilled practitioners will be capable of selecting suitablevectors and mammalian cells for introducing any of spatial profilingreagents described herein.

9. Other Methods and Cell-Tagging Agents for Intracellular Introductionof a Molecule

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by the use of a needle, for example for injection(e.g., microinjection), particle bombardment, photoporation,magnetofection, and/or hydroporation. For example, with particlebombardment, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) and a capture domain can be coated with heavymetal particles and delivered to a cell at a high speed. Inphotoporation, a transient pore in a cell membrane can be generatedusing a laser pulse, allowing cellular uptake of a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain. In magnetofection, a molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and a capturedomain can be coupled to a magnetic particle (e.g., magneticnanoparticle, nanowires, etc.) and localized to a target cell via anapplied magnetic field. In hydroporation, a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain can be delivered to a cell and/or cell bead via hydrodynamicpressure.

(iii) Lipid Tagged Primers/Lipophilic-Tagged Moieties

In some embodiments of any of the spatial profiling methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) is coupled to a lipophilic molecule. In someembodiments, the lipophilic molecule enables delivery of the lipophilicmolecule to the cell membrane or the nuclear membrane. In someembodiments, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) coupled to a lipophilic molecule can associatewith and/or insert into lipid membranes such as cell membranes andnuclear membranes. In some cases, the insertion can be reversible. Insome cases, the association between the lipophilic molecule and the cellmay be such that the cell retains the lipophilic molecule (e.g., andassociated components, such as nucleic acid barcode molecules) duringsubsequent processing (e.g., partitioning, cell permeabilization,amplification, pooling, etc.). In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode)coupled to a lipophilic molecule may enter into the intracellular spaceand/or a cell nucleus.

Non-limiting examples of lipophilic molecules that can be used inembodiments described herein include sterol lipids such as cholesterol,tocopherol, steryl, palmitate, lignoceric acid, and derivatives thereof.In some embodiments, the lipophilic molecules are neutral lipids thatare conjugated to hydrophobic moieties (e.g., cholesterol, squalene, orfatty acids) (See Raouane et al. Bioconjugate Chem., 23(6):1091-1104(2012) which is herein incorporated by reference in its entirety). Insome embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) may be attached to the lipophilicmoiety via a linker, using covalent or direct attachment. In someembodiments, the linker is a tetra-ethylene glycol (TEG) linker. Otherexemplary linkers include, but are not limited to, Amino Linker C6,Amino Linker C12, Spacer C3, Spacer C6, Spacer C12, Spacer 9, and Spacer18. In some embodiments, a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) is indirectly coupled (e.g.,via hybridization or ligand-ligand interactions, such asbiotin-streptavidin) to a lipophilic molecule. In some embodiments, thelipophilic moiety can be attached to a capture probe, spatial barcode,or other DNA sequence, at either the 5′ or 3′ end of the specified DNAsequence. In some embodiments, the lipophilic moiety can be coupled to acapture probe, spatial barcode, or other DNA sequence in alipid-dependent manner. Other lipophilic molecules that may be used inaccordance with methods provided herein include amphiphilic moleculeswherein the headgroup (e.g., charge, aliphatic content, and/or aromaticcontent) and/or fatty acid chain length (e.g., C12, C14, C16, or C18)can be varied. For instance, fatty acid side chains (e.g., C12, C14,C16, or C18) can be coupled to glycerol or glycerol derivatives (e.g.,3-t-butyldiphenylsilylglycerol), which can also comprise, e.g., acationic head group. In some embodiments, a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) disclosedherein can then be coupled (either directly or indirectly) to theseamphiphilic molecules. In some embodiments, a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) coupled to anamphiphilic molecule may associate with and/or insert into a membrane(e.g., a cell, cell bead, or nuclear membrane). In some cases, anamphiphilic or lipophilic moiety may cross a cell membrane and provide amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) to an internal region of a cell and/or cell bead.

In some embodiments, additives can be added to supplement lipid-basedmodifications. In some embodiments, the additive is low densitylipoprotein (LDL). In some embodiments, the additive is the cholesteroltrafficking inhibitor U-18666A. In some embodiments, U-18666A inhibitscholesterol transport from late endosomes at micromolar concentrationsand/or lysosomes to the endoplasmic reticulum (ER) at nanomolarconcentrations. In some embodiments, U-18666A can inhibit oxidosqualenecyclase, a key enzyme in the cholesterol biosynthesis pathway, atsufficiently high concentrations (e.g., at or about >0.5 mM).

In some embodiments, wherein the molecule (e.g., with a nucleic acidsequence) has an amino group within the molecule, the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and anamino group can be coupled to an amine-reactive lipophilic molecule. Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) and an amino group can be conjugated toDSPE-PEG(2000)-cyanuric chloride(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethyleneglycol)-2000]).

In some embodiments, a cell-tagging agent can attach to a surface of acell through a combination of lipophilic and covalent attachment. Forexample, a cell-tagging agent can include an oligonucleotide attached toa lipid to target the oligonucleotide to a cell membrane, and an aminegroup that can be covalently linked to a cell surface protein(s) via anynumber of chemistries described herein. In these embodiments, the lipidcan increase the surface concentration of the oligonucleotide and canpromote the covalent reaction.

As used herein, an “anchor oligonucleotide” and/or “co-anchoroligonucleotide” can include a lipid-conjugated oligonucleotide, whereinthe lipid is capable of becoming embedded within a cell membrane. Insome embodiments, the lipid capable of becoming embedded within a cellmembrane includes but is not limited to, sterol lipids such ascholesterol, tocopherol, steryl, palmitate, lignoceric acid, andderivatives thereof. In some embodiments, the sterol lipid of the anchoroligonucleotide and/or co-anchor oligonucleotide can be attached toeither the 5′ or 3′ end of the oligonucleotide portion. In someembodiments, the anchor oligonucleotide and/or the co-anchoroligonucleotide can integrate into the cell membrane of a cell in abiological sample (e.g., the sterol lipid of the anchor oligonucleotideand/or co-anchor oligonucleotide).

In some embodiments, a sterol lipid (e.g., lignoceric acid) anchoroligonucleotide is attached to the 5′ end of the oligonucleotide. Insome embodiments, the anchor oligonucleotide can have a constantsequence. In some embodiments the constant sequence of the anchoroligonucleotide can be about 15 to about 30 nucleotides long. In someembodiments, the anchor oligonucleotide can have an additional domain 3′to the constant sequence. In some embodiments, the additional domain canbe an adapter sequence (e.g., sequencing adapter). In some embodiments,the adapter sequence can be about 15 to about 35 nucleotides long.

In some embodiments, the lipid (e.g., sterol lipid) of the co-anchoroligonucleotide (e.g., palmitic acid), is attached to the 3′ end of theoligonucleotide. In some embodiments, the co-anchor oligonucleotide canhave a constant sequence. For example, the constant sequence of theco-anchor oligonucleotide can be a reverse complement of the constantsequence from the anchor oligonucleotide. In some embodiments, theconstant sequence of the anchor oligonucleotide and the constantsequence of the co-anchor oligonucleotide can bind (e.g., hybridize) toeach other. In some embodiments, the lipid (e.g., sterol lipid) of theanchor oligonucleotide and the co-anchor oligonucleotide can integrateinto a cell membrane in the biological sample and the respectiveconstant sequences can hybridize to each other at the same time. In someembodiments, a barcoded oligonucleotide, which can include severaldomains, can be introduced to the integrated anchor oligonucleotide andco-anchor oligonucleotide hybridized to each other. The barcodedoligonucleotide can include, in a 5′ to 3′ direction, a functionaldomain (e.g., a sequencing adapter domain), a unique molecularidentifier, a sample barcode, a second unique molecular identifier, andthe reverse complement of a constant sequence. For example, aftertagging a cell with any of the cell-tagging agents described herein thecells can be partitioned (e.g., encapsulated in a vesicle) with abarcoded feature (e.g., a bead). In some embodiments, the reversecomplement of the constant sequence of the barcoded oligonucleotide caninteract (e.g., hybridize) with the constant sequence (e.g., a portionof the sequence) on the barcoded feature.

(iv) Intracellular Cleavage Domain

As used herein, capture probes can optionally include an “intracellularcleavage domain,” wherein one or more segments or regions of the captureprobe (e.g., capture domains, spatial barcodes, and/or UMIs) can bereleasably or cleavably attached to one or more other segments orregions of the capture probe, such as a cell-penetrating agent, suchthat the capture domain, spatial barcode, and/or UMI can be released orbe releasable through cleavage of a linkage between the capture domain,spatial barcode, and/or UMI and the cell-penetrating agent and/or cellpenetration tag. In some embodiments, the cleavage of the linkagebetween the capture domain, spatial barcode, and/or UMI and thecell-penetrating agent is induced in an intracellular environment (e.g.,the intracellular cleavage domain is cleaved after the capture probes isintroduced into the cell). For example, the linkage between the capturedomain, spatial barcode, and/or UMI and the cell-penetrating agent canbe a disulfide bond that is cleaved by the reducing conditions in thecell, for example, when the intracellular cleavage domain comprises adisulfide bond. Any other suitable linker can be used to release orcleave the intracellular cleavage domain of the capture probe.

(v) Positive or Neutral Oligo-Conjugated Polymers

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a glycol chitosanderivative. In some embodiments, the glycol chitosan derivative can becoupled with two or more molecules (e.g., nucleic acid molecules) havinga barcode (e.g., a spatial barcode). In some embodiments, the glycolchitosan derivative can be coupled with about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10 or more molecules. The glycolchitosan derivative (e.g., glycol chitosan-cholesterol) can serve as ahydrophobic anchor (see Wang et al. J. Mater. Chem. B., 30:6165 (2015),which is herein incorporated by reference in its entirety). Non-limitingexamples of chitosan derivatives that can be coupled to a molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) can be found in Cheung et al., Marine Drugs, 13(8): 5156-5186(2015), which is herein incorporated by reference in its entirety.

(vi) Bifunctional NHS Linker Cell-Tagging

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a bifunctional NHS linker.In some embodiments, the coupled bifunctional NHS linker (e.g.,bifunctional linker and the molecule having a barcode) can facilitatethe attachment of the spatial barcode to the surface of the cell. Insome embodiments, after facilitating attachment to the surface of thecell, excess NHS linker can be removed (e.g., washed away). In someembodiments, the process of coupling the molecule having a barcode canbe performed under non-anhydrous conditions to maintain the activity ofunreacted bifunctional NHS. In some embodiments, the non-anhydrouscondition can be in the presence of DMSO. In some embodiments, thenon-anhydrous condition can be in the presence of DMF.

(vii) Antibody-Tagged Primers

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to an antibody or antigenbinding fragment thereof in a manner that facilitates attachment of themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) to the surface of a cell. In some embodiments,facilitating attachment to the cell surface facilitates introduction ofthe molecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) into the cell. In some embodiments, the molecule (e.g.,a nucleic acid molecule) having a barcode (e.g., a spatial barcode) canbe coupled to an antibody that is directed to an antigen that is presenton the surface of a cell. In some embodiments, the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) can becoupled to an antibody that is directed to an antigen that is present onthe surface of a plurality of cells (e.g., a plurality of cells in abiological sample). In some embodiments, the molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) can be coupledto an antibody that is directed to an antigen that is present on thesurface of a cell, a plurality of cells, or substantially all the cellspresent in a biological sample. In some embodiments, thebarcoded-antibody is directed to an intracellular antigen. Any of theexemplary methods described herein of attaching a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) toanother molecule (e.g., an antibody or antigen fragment thereof) can beused.

(viii) Streptavidin-Conjugated Oligonucleotides

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can attach to the surface of a cell usingbiotin-streptavidin. In some embodiments, primary amines in the sidechain of lysine residues of cell surface polypeptides are labelled withNHS-activated biotin reagents. For example, the N-terminus of apolypeptide can react with NHS-activated biotin reagents to form stableamide bonds. In some embodiments, cell-tagging agents include molecules(e.g., a nucleic acid molecule) having barcodes (e.g., a spatialbarcode) conjugated to streptavidin. In some cases, streptavidin can beconjugated to the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) using click chemistry (e.g., maleimidemodification) as described herein. In some embodiments, a cellcontaining NHS-activated biotin incorporated into lysine side chains ofa cell surface protein forms a non-covalent bond with the streptavidinconjugated to the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode). In some embodiments, the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) conjugated to streptavidin is itself part of a cell-taggingagent.

(ix) Dye-Tagged Oligonucleotides

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) is directly linked to a detectable label. Insome embodiments, the detectable label is any of the detectable labelsdescribed herein. In some embodiments the detectable label is afluorescent tag. In some embodiments, the physical properties of thefluorescent tags (e.g., a fluorescent tag having hydrophobic properties)can overcome the hydrophilic nature of the molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode). For example,in some embodiments, wherein the molecule is a nucleic acid molecule, afluorescent tag (e.g., BODIPY, Cy3, Atto 647N, and Rhodamine Red C2) canbe coupled to a 5′ end of the nucleic acid molecule having a barcode(e.g., a spatial barcode). In some embodiments, wherein the molecule isa nucleic acid molecule, any fluorescent tag having hydrophobicproperties can be coupled to the nucleic acid molecule having a barcode(e.g., a spatial barcode) in a manner that overcomes the hydrophilicnature of the nucleic acid molecule. Non-limiting examples offluorescent tags with hydrophobic properties include BODIPY, Cy3, Atto647N, and Rhodamine Red C2.

(x) Click-Chemistry

In some embodiments of any of the spatial analysis methods describedherein, molecules (e.g., a nucleic acid molecule) having barcodes (e.g.,a spatial barcode) are coupled to click-chemistry moieties. As usedherein, the term “click chemistry,” generally refers to reactions thatare modular, wide in scope, give high yields, generate byproducts, suchas those that can be removed by nonchromatographic methods, and arestereospecific (but not necessarily enantioselective) (see, e.g., Angew.Chem. Int. Ed., 2001, 40(11):2004-2021, which is incorporated herein byreference in its entirety). In some cases, click chemistry can describepairs of functional groups that can selectively react with each other inmild, aqueous conditions.

An example of a click chemistry reaction is the Huisgen 1,3-dipolarcycloaddition of an azide and an alkyne, i.e., copper-catalyzed reactionof an azide with an alkyne to form the 5-membered heteroatom ring1,2,3-triazole. The reaction is also known as a Cu(I)-CatalyzedAzide-Alkyne Cycloaddition (CuAAC), a Cu(I) click chemistry or a Cu+click chemistry. Catalysts for the click chemistry include, but are notlimited to, Cu(I) salts, or Cu(I) salts made in situ by reducing Cu(II)reagents to Cu(I) reagents with a reducing reagent (Pharm Res. 2008,25(10): 2216-2230, which is incorporated herein by reference in itsentirety). Known Cu(II) reagents for the click chemistry can include,but are not limited to, the Cu(II)-(TBTA) complex and the Cu(II) (THPTA)complex. TBTA, which istris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known astris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I)salts. THPTA, which is tris-(hydroxypropyltriazolylmethyl)amine, isanother example of a stabilizing agent for Cu(I). Other conditions canalso be used to construct the 1,2,3-triazole ring from an azide and analkyne using copper-free click chemistry, such as the Strain-promotedAzide-Alkyne Click chemistry reaction (SPAAC) (see, e.g., Chem. Commun.,2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90, each of which isincorporated herein by reference in its entirety).

In some embodiments of any of the spatial analysis described herein,molecules (e.g., a nucleic acid molecule) having barcodes (e.g., aspatial barcode) are coupled to click-chemistry moieties without copperbeing present (e.g., copper-free click chemistry). One exemplarycopper-free click chemistry methodology comprises a reaction betweencyclooctyne and phenyl azide that results in the product1-phenyl-4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazole (See,e.g., 2010, Akeroyd, N., et al., Click chemistry for the preparation ofadvance macromolecular architectures, PhD dissertation, incorporatedherein by reference in its entirety.). Additional copper-free clickchemistry methods are known to those skilled in the art (See, e.g.,2009, Click Chemistry for Biotechnology and Materials Science, Ed. JeorgLahann, John Wiley & Sons, Ltd. publ., p. 410).

(xi) Receptor-Ligand Systems

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a ligand, wherein the ligandis part of a receptor-ligand interaction on the surface of a cell. Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a ligand that interactsselectively with a cell surface receptor thereby targeting the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) to a specific cell. Non-limiting examples of receptor-ligandsystems that can be used include integrin receptor-ligand interactions,GPCR receptor-ligand interactions, RTK receptor-ligand interactions, andTLR-ligand interactions (see Juliano, Nucleic Acids Res., 44(14):6518-6548 (2016), which is incorporated herein by reference in itsentirety). Any of the methods described herein for attaching a molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) to a ligand (e.g., any of the methods described herein relatingto attaching a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) to an antibody) can be used.

(xii) Covalent Binding Systems via Amine or Thiol Functionalities

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can incorporate reactive functional groups atsites within the molecule (e.g., with a nucleic acid sequence). In suchcases, the reactive functional groups can facilitate conjugation toligands and/or surfaces. In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) caninclude thiol modifiers that are designed to react with a broad array ofactivated accepting groups (e.g., maleimide and gold microspheres). Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) having thiol modifiers can interact with amaleimide-conjugated peptide thereby resulting in labelling of thepeptide. In some embodiments, maleimide-conjugated peptides are presenton the surface of a cell whereupon interaction with the thiol-modifiedmolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode), the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) is coupled to the surface of the cell.Non-limiting examples of thiol modifiers include: 5′ thiol modifier C6S—S, 3′ thiol modifier C3 S—S, dithiol, 3′ thiol modifier oxa 6-S—S, anddithiol serinol.

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can include amine modifiers, e.g., aminemodifiers that are designed to attach to another molecule in thepresence of an acylating agent. In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) caninclude amine modifiers that are designed to attach to a broad array oflinkage groups (e.g., carbonyl amide, thiourea, sulfonamide, andcarboxamide). For example, a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) and an amine modifier caninteract with a sulfonamide-conjugated peptide thereby resulting inlabelling of the peptide. In some embodiments, sulfonamide-conjugatedpeptides are present on the surface of a cell whereupon interaction withthe amine-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode), the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) is coupled to thesurface of the cell. Non-limiting example of amine modifiers include:DMS(O)MT-Amino-Modifier-C6, Amino-Modifier-C3-TFA, Amino-Modifier-C12,Amino-Modifier-C6-TFA, Amino-dT, Amino-Modifier-5, Amino-Modifier-C2-dT,Amino-Modifier-C6-dT, and 3′-Amino-Modifier-C7.

As another example, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can incorporate reactive functionalgroups at sites within the molecule (e.g., with a nucleic acid sequence)such as N-hydroxysuccinimide (NHS). In some embodiments, amines (e.g.,amine-containing peptides) are present on the surface of a cellwhereupon interaction with the NHS-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode), the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) is coupled to the surface of the cell. In some embodiments, amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) is reacted with a bifunctional NHS linker to form anNHS-modified molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode).

In some embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can be coupled to a biocompatibleanchor for cell membrane (BAM). For example, a BAM can include moleculesthat comprise an oleyl group and PEG. The oleyl group can facilitateanchoring the molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) to a cell, and the PEG can increase watersolubility. In some embodiments, oleyl-PEG-NHS can be coupled to amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) using NHS chemistry.

(xiii) Azide-Based Systems

In some embodiments, wherein the molecule (e.g., with a nucleic acidsequence) incorporates reactive functional groups at sites within themolecule, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to an azide group on a cellsurface. In some embodiments, the reactive functional group is analkynyl group. In some embodiments, click chemistry as described hereincan be used to attach the alkynyl-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) to an azidegroup on the cell surface. An azide group can be attached to the cellsurface through a variety of methods. For example, NHS chemistry can beused to attach an azide group to the cell surface. In some embodiments,N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), which contains anazide group, can react with sialic acid on the surface of a cell toattach azide to the cell surface. In some embodiments, azide is attachedto the cell surface by bio-orthogonal expression of azide. For example,azide is incubated with the cells. In some embodiments, thealkynyl-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can attach to the surface of a cellvia an azide group in the presence of copper. In some embodiments, thealkynyl-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can attach to the surface of a cellvia an azide group in the absence of copper.

(xiv) Lectin-Based Systems

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a lectin that facilitatesattachment of the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) to a cell surface. Lectin can bind toglycans, e.g., glycans on the surface of cells. In some embodiments, themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) has an incorporated reactive functional group such asan azide group. In some embodiments, the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and an azide groupis reacted with a modified lectin, e.g., a lectin modified using NHSchemistry to introduce an azide reactive group. In some embodiments, alive cell is labelled with a lectin-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode). In someembodiments, a fixed cell is labelled with a lectin-modified molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode). In some embodiments, a permeabilized cell is labelled with alectin-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode). In some embodiments, organelles inthe secretory pathway can be labelled with a lectin-modified molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode).

(b) Methods for Separating a Sample into Single Cells or Cell Groups

Some embodiments of any of the methods described herein can includeseparating a biological sample into single cells, cell groups, types ofcells, or a region or regions of interest. For example, a biologicalsample can be separated into single cells, cell groups, types of cells,or a region or regions of interest before being contacted with one ormore capture probes. In other examples, a biological sample is firstcontacted with one or more capture probes, and then separated intosingle cells, cell groups, types of cells, or a region or regions ofinterest.

In some embodiments, a biological sample can be separated into chucksusing pixelation. Pixelation can include the steps of providing abiological sample, and punching out one or more portions of thebiological sample. The punched out portions of the biological sample canthen be used to perform any of the methods described herein. In someembodiments, the punched-out portions of the biological sample can be ina random pattern or a designed pattern. In some embodiments, thepunched-out portions of the biological sample can be focused on a regionof interest or a subcellular structure in the biological sample.

FIG. 20A is a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for “pixelating” a sample, wherein the sample iscut, stamped, microdissected, or transferred by hollow-needle ormicroneedle, moving a small portion of the sample into an individualpartition or well.

FIG. 20B is a schematic depicting multi-needle pixelation, wherein anarray of needles is punched through a sample on a scaffold, intonanowells containing beads (e.g., gel beads) and reagents. Once theneedle is in the nanowell, the cell(s) are ejected.

In some embodiments, a biological sample (e.g., a tissue sample ortissue section) is divided into smaller portions as compared to theoriginal biological sample size (“chunks”) before performance of any ofthe spatial analysis methods described herein. In some embodiments, themethods can include spatial barcoding of FFPE “chunks” via barcodesapplied in a spatially well-defined pattern (e.g., array printing). Inorder to associate a spatial barcode with a particular “chunk” ofbiological sample, the barcode (e.g., a spatial barcode) can be of asufficient length to prevent diffusion of the barcode in subsequentsteps, or the spatial barcode can be covalently applied to the FFPEsample. In some embodiments, the spatial barcode is unique to each FFPEchunk. In some embodiments, spatial barcodes can be embedded onto anFFPE slide (e.g., within a matrix, such as a wax or a hydrogel). In someembodiments, the FFPE slide is heated (e.g., wax is heated) prior toaddition of the spatial barcodes. In some embodiments, after addition ofthe spatial barcodes, the FFPE slide can be cooled and cut ordissociated into chunks. Methods of chunking (e.g., cutting) biologicalsamples are known in the art. For example, in a non-limiting example,chunking of biological samples can be done in various ways such as lasermicrodissection, mechanical means, acoustic (e.g., sonication) means, orany other method described herein. In some embodiments,fluorophores/Qdots, etc. can be embedded in the chunk to preservespatial information about the biological sample. Barcoding at this stepenables massively parallel encapsulation of chunks while retaining localspatial information (e.g., tumor versus normal/healthy cells). In someembodiments, chunking of a biological sample (e.g., a tissue section)can result in single-cell chunks of the biological sample. In otherembodiments, chunking of a biological sample can be performed to obtainchunks that correspond to diseased portions of the biological sample. Inanother embodiment, chunking of biological samples can be performed toobtain discrete chunks of the biological sample that correspond todiseased or healthy portions of the biological sample. In someembodiments, chunking of biological samples can be performed to obtainchunks that correspond to specific cell types (e.g., chunking based onfluorescent or chemiluminescent imaging of antibodies bound to targetproteins) in the biological sample.

In some embodiments, the spatially-barcoded chunks can be furtherprocessed. For example, the spatially-barcoded chunk can be individuallyencapsulated (e.g., a matrix, emulsion, or hydrogel). In someembodiments, the spatially-barcoded chunk can be encapsulated in apartition (e.g., a well, droplet, channel, or vesicle). In someembodiments, the spatially-barcoded chunk can be encapsulated in avesicle. In some embodiments, the vesicle can comprise a lipid bilayer.In some embodiments, the spatially-barcoded FFPE chunk can beencapsulated with a uniquely barcoded bead. In some embodiments, theuniquely barcoded bead can have a functional domain, a cleavage domain,a unique molecular identifier, and a capture domain, or combinationsthereof. In some embodiments, the encapsulated spatially-barcoded FFPEchunk and the uniquely barcoded bead can be heated to deparaffinize theFFPE sample. In some embodiments, the encapsulated spatially-barcodedFFPE chunk and the uniquely barcoded bead can be treated with xylene todeparaffinize the FFPE sample. In some embodiments, the deparaffinizedsample can be treated to de-crosslink methylene bridges in a singlestep. In some embodiments, additional steps can be performed when, forexample, de-crosslinking chemistry is incompatible with barcoding orlibrary preparation steps. In some embodiments, after de-crosslinkingmethylene bridges, the nucleic acids originating or present in the chunkcan bind to the uniquely barcoded bead. In some embodiments, after thespatial barcode binds the uniquely barcoded bead, the encapsulation canbe disrupted (e.g., lysed, melted, or removed) and the barcoded beadscan be collected. In some embodiments, the collected barcoded beads canbe washed and re-encapsulated. In some embodiments, the nucleic acidsassociated with the bead (e.g., spatial barcode, unique barcode, analytetranscript) can be amplified (e.g., PCR amplified) and processed (e.g.,sequenced) according to any of the methods described herein.

In some embodiments, a biological sample can be divided or portionedusing laser capture microdissection (e.g., highly-multiplexed lasercapture microdissection).

(c) Release and Amplification of Analytes

In some embodiments, lysis reagents can be added to the sample tofacilitate release of analyte(s) from a sample. Examples of lysis agentsinclude, but are not limited to, bioactive reagents such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other commercially available lysis enzymes. Other lysisagents can additionally or alternatively be co-partitioned with thebiological sample to cause the release of the sample's contents into thepartitions. In some embodiments, surfactant-based lysis solutions can beused to lyse cells, although these can be less desirable foremulsion-based systems where the surfactants can interfere with stableemulsions. Lysis solutions can include ionic surfactants such as, forexample, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation,thermal, acoustic or mechanical cellular disruption can also be used incertain embodiments, e.g., non-emulsion based partitioning such asencapsulation of biological materials that can be in addition to or inplace of droplet partitioning, where any pore volume of the encapsulateis sufficiently small to retain nucleic acid fragments of a given size,following cellular disruption.

In addition to the permeabilization agents, other reagents can also beadded to interact with the biological sample, including, for example,DNase and RNase inactivating agents or inhibitors or chelating agents,such as EDTA, and other reagents to allow for subsequent processing ofanalytes from the sample. In other embodiments, nucleases, such as DNaseor RNAse, or proteases, such as pepsin or proteinase K, are added to thesample.

Further reagents that can be added to a sample, include, for example,endonucleases to fragment DNA, DNA polymerase enzymes, and dNTPs used toamplify nucleic acids. Other enzymes that can also be added to thesample include, but are not limited to, polymerase, transposase, ligase,proteinase K, and DNAse, etc. Additional reagents can also includereverse transcriptase enzymes, including enzymes with terminaltransferase activity, primers, and switch oligonucleotides. In someembodiments, template switching can be used to increase the length of acDNA, e.g., by appending a predefined nucleic acid sequence to the cDNA.

If a tissue sample is not permeabilized sufficiently, the amount ofanalyte captured on the substrate can be too low to enable adequateanalysis. Conversely, if the tissue sample is too permeable, the analytecan diffuse away from its origin in the tissue sample, such that therelative spatial relationship of the analytes within the tissue sampleis lost. Hence, a balance between permeabilizing the tissue sampleenough to obtain good signal intensity while still maintaining thespatial resolution of the analyte distribution in the tissue sample isdesired.

In some embodiments, where the biological sample includes live cells,permeabilization conditions can be modified so that the live cellsexperience only brief permeabilization (e.g., through short repetitivebursts of electric field application), thereby allowing one or moreanalytes to migrate from the live cells to the substrate while retainingcellular viability. In some embodiments, after contacting a biologicalsample with a substrate that include capture probes, a removal step isperformed to remove all or a portion of the biological sample from thesubstrate. In some embodiments, the removal step includes enzymatic orchemical degradation of the permeabilized cells of the biologicalsample. For example, the removal step can include treating thebiological samples with an enzyme (e.g., proteinase K) to remove atleast a portion of the biological sample from the first substrates. Insome embodiments, the removal step can include ablation of the tissue(e.g., laser ablation).

In some embodiments, where RNA is captured from cells in a sample, oneor more RNA species of interest can be selectively enriched. Forexample, one or more species of RNA of interest can be selected byaddition of one or more oligonucleotides. One or more species of RNA canbe selectively down-selected (e.g., removed) using any of a variety ofmethods. For example, probes can be administered to a sample thatselectively hybridize to ribosomal RNA (rRNA), thereby reducing the pooland concentration of rRNA in the sample. Subsequent application of thecapture probes to the sample can result in improved RNA capture due tothe reduction in non-specific RNA present in the sample. In someembodiments, the additional oligonucleotide is a sequence used forpriming a reaction by a polymerase. For example, one or more primersequences with sequence complementarity to one or more RNAs of interest,can be used to amplify the one or more RNAs of interest, therebyselectively enriching these RNAs. In some embodiments, anoligonucleotide with sequence complementarity to the complementarystrand of captured RNA (e.g., cDNA) can bind to the cDNA. In onenon-limiting example, biotinylated oligonucleotides with sequencecomplementary to one or more cDNA of interest binds to the cDNA and canbe selected using biotinylation-strepavidin affinity in any number ofmethods known to the field (e.g., streptavidin beads).

In some embodiments, any of the spatial analysis methods describedherein can include modulating the rate of interaction between biologicalanalytes from the biological sample and the capture probes on the array.In some embodiments, modulating the rate of interaction can occur bymodulating the biological sample (e.g., modulating temperature or pH).In some embodiments, modulating the rate of interaction includes usingexternal stimuli. Non-limiting examples of external stimuli that can beused to modulate the rate of interaction include light, temperature,small molecules, enzymes, and/or an activating reagent. In one example,light can be used to activate a polymerase in a nucleic acid extensionreaction. In another example, temperature can be used to modulatehybridization between two complementary nucleic acid molecules.

Nucleic acid analytes can be amplified using a polymerase chain reaction(e.g., digital PCR, quantitative PCR, or real time PCR), isothermalamplification, or any nucleic acid amplification or extension reactionsdescribed herein, or known in the art.

(d) Partitioning

As discussed above, in some embodiments, the sample can optionally beseparated into single cells, cell groups (e.g., based on cell sub-typeor gene expression profile), or other fragments/pieces that are smallerthan the original sample. Each of these smaller portions of the samplecan be analyzed to obtain spatially-resolved analyte information fromthe sample. Non-limiting partitioning methods are described herein.

For samples that have been separated into smaller fragments—andparticularly, for samples that have been disaggregated, dissociated, orotherwise separated into individual cells—one method for analyzing thefragments involves partitioning the fragments into individual partitions(e.g., fluid droplets), and then analyzing the contents of thepartitions. In general, each partition maintains separation of its owncontents from the contents of other partitions. For example, thepartition can be a droplet in an emulsion.

The methods described herein provide for the compartmentalization orpartitioning of a cell (e.g., a cell) from a sample into discretecompartments or voxels. As used herein, each “voxel” represents a3-dimensional volumetric unit. In some embodiments, a voxel maintainsseparation of its own contents from the contents of other voxels. Avoxel can be one partition of an array of partitions of volume. Forexample, a voxel can be one partition of an array of discrete partitionsinto which a 3-dimensional object is divided. As another example,members of a plurality of photo-crosslinkable polymer precursors can becross-linked into voxels that are part of an array of thephoto-crosslinked polymer covering the substrate or a portion of thesubstrate. Unique identifiers, e.g., barcodes, may be previously,subsequently, or concurrently delivered to the cell, in order to allowfor the later attribution of the characteristics of the cell to theparticular voxel. In some embodiments, a voxel has defined dimensions.In some embodiments, a voxel comprises a single cell.

For example, a substrate can be coated with a DTT-sensitive hydrogel andthen contacted with a biological sample. Optionally, capture probesattached to the substrate are released from the substrate such that thereleased capture probes are introduced into the biological sample and atleast one released capture probe interacts with at least one biologicalanalyte present in the biological sample via the capture domain. Thebiological sample and substrate can be assembled into a flow-cell and aphoto-crosslinkable polymer precursor added. The cells of the biologicalsample can be then crosslinked into hydrogel-voxels of defineddimensions using a light source. The flow-cell can be dismantled andwashed to remove unpolymerized polymer precursors. The coating can betreated with DTT to yield single-cell partitions for use in downstreamapplications. The capture probes/biological analytes can be analyzed,and the spatial information of the spatially-barcoded features can beused to determine the spatial location of the captured biologicalanalytes in the biological sample.

In addition to analytes, a partition can include additional components,and in particular, one or more beads. A partition can include a singlegel bead, a single cell bead, or both a single cell bead and single gelbead.

A partition can also include one or more reagents. Unique identifiers,such as barcodes, can be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead). Microfluidic channel networks (e.g., on achip) can be utilized to generate partitions. Alternative mechanisms canalso be employed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions caninclude, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionscan include a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions can beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, the entire contents of whichare incorporated herein by reference. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are described,for example, in U.S. Patent Application Publication No. 2010/0105112,the entire contents of which are incorporated herein by reference.

For droplets in an emulsion, allocating individual particles to discretepartitions can be accomplished, for example, by introducing a flowingstream of particles in an aqueous fluid into a flowing stream of anon-aqueous fluid, such that droplets are generated at the junction ofthe two streams. Fluid properties (e.g., fluid flow rates, fluidviscosities, etc.), particle properties (e.g., volume fraction, particlesize, particle concentration, etc.), microfluidic architectures (e.g.,channel geometry, etc.), and other parameters can be adjusted to controlthe occupancy of the resulting partitions (e.g., number of analytes perpartition, number of beads per partition, etc.) For example, partitionoccupancy can be controlled by providing the aqueous stream at a certainconcentration and/or flow rate of analytes.

To generate single analyte partitions, the relative flow rates of theimmiscible fluids can be selected such that, on average, the partitionscan contain less than one analyte per partition to ensure that thosepartitions that are occupied are primarily singly occupied. In somecases, partitions among a plurality of partitions can contain at mostone analyte. In some embodiments, the various parameters (e.g., fluidproperties, particle properties, microfluidic architectures, etc.) canbe selected or adjusted such that a majority of partitions are occupied,for example, allowing for only a small percentage of unoccupiedpartitions. The flows and channel architectures can be controlled as toensure a given number of singly occupied partitions, less than a certainlevel of unoccupied partitions and/or less than a certain level ofmultiply occupied partitions.

The channel segments described herein can be coupled to any of a varietyof different fluid sources or receiving components, includingreservoirs, tubing, manifolds, or fluidic components of other systems.As will be appreciated, the microfluidic channel structure can have avariety of geometries. For example, a microfluidic channel structure canhave one or more than one channel junction. As another example, amicrofluidic channel structure can have 2, 3, 4, or 5 channel segmentseach carrying particles that meet at a channel junction. Fluid can bedirected to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can include compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid can also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary, and/or gravity flow.

A partition can include one or more unique identifiers, such asbarcodes. Barcodes can be previously, subsequently, or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes can be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes canbe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can include a bead.

In some embodiments, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus candisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

In some embodiments, one more barcodes (e.g., spatial barcodes, UMIs, ora combination thereof) can be introduced into a partition as part of theanalyte. As described previously, barcodes can be bound to the analytedirectly, or can form part of a capture probe or analyte capture agentthat is hybridized to, conjugated to, or otherwise associated with ananalyte, such that when the analyte is introduced into the partition,the barcode(s) are introduced as well.

FIG. 21 depicts an exemplary workflow, where a sample is contacted witha spatially-barcoded capture probe array and the sample is fixed,stained, and imaged 2101, as described elsewhere herein. The captureprobes can be cleaved from the array 2102 using any method as describedherein. The capture probes can diffuse toward the cells by eitherpassive or active migration as described elsewhere herein. The captureprobes may then be introduced to the sample 2103 as described elsewhereherein, wherein the capture probe is able to gain entry into the cell inthe absence of cell permeabilization, using one of the cell penetratingpeptides or lipid delivery systems described herein. The sample can thenbe optionally imaged in order to confirm probe uptake, via a reportermolecule incorporated within the capture probe 2104. The sample can thenbe separated from the array and undergo dissociation 2105, wherein thesample is separated into single cells or small groups of cells. Once thesample is dissociated, the single cells can be introduced to an oil-inwater droplet 2106, wherein a single cell is combined with reagentswithin the droplet and processed so that the spatial barcode thatpenetrated the cell labels the contents of that cell within the droplet.Other cells undergo separately partitioned reactions concurrently. Thecontents of the droplet is then sequenced 2107 in order to associate aparticular cell or cells with a particular spatial location within thesample 2108.

As described above, FIG. 16 shows an example of a microfluidic channelstructure for partitioning individual analytes (e.g., cells) intodiscrete partitions. FIGS. 17A and 17C also show other examples ofmicrofluidic channel structures that can be used for delivering beads todroplets.

A variety of different beads can be incorporated into partitions asdescribed above. In some embodiments, for example, non-barcoded beadscan be incorporated into the partitions. For example, where thebiological particle (e.g., a cell) that is incorporated into thepartitions carries one or more barcodes (e.g., spatial barcode(s),UMI(s), and combinations thereof), the bead can be a non-barcoded bead.

In some embodiments, a barcode carrying bead can be incorporated intopartitions. For example, a nucleic acid molecule, such as anoligonucleotide, can be coupled to a bead by a releasable linkage, suchas, for example, a disulfide linker. The same bead can be coupled (e.g.,via releasable linkage) to one or more other nucleic acid molecules. Thenucleic acid molecule can be or include a barcode. As noted elsewhereherein, the structure of the barcode can include a number of sequenceelements.

The nucleic acid molecule can include a functional domain that can beused in subsequent processing. For example, the functional domain caninclude one or more of a sequencer specific flow cell attachmentsequence (e.g., a P5 sequence for Illumina® sequencing systems) and asequencing primer sequence (e.g., a R1 primer for Illumina® sequencingsystems). The nucleic acid molecule can include a barcode sequence foruse in barcoding the sample (e.g., DNA, RNA, protein, etc.). In somecases, the barcode sequence can be bead-specific such that the barcodesequence is common to all nucleic acid molecules coupled to the samebead. Alternatively or in addition, the barcode sequence can bepartition-specific such that the barcode sequence is common to allnucleic acid molecules coupled to one or more beads that are partitionedinto the same partition. The nucleic acid molecule can include aspecific priming sequence, such as an mRNA specific priming sequence(e.g., poly (T) sequence), a targeted priming sequence, and/or a randompriming sequence. The nucleic acid molecule can include an anchoringsequence to ensure that the specific priming sequence hybridizes at thesequence end (e.g., of the mRNA). For example, the anchoring sequencecan include a random short sequence of nucleotides, such as a 1-mer,2-mer, 3-mer or longer sequence, which can ensure that a poly(T) segmentis more likely to hybridize at the sequence end of the poly(A) tail ofthe mRNA.

The nucleic acid molecule can include a unique molecular identifyingsequence (e.g., unique molecular identifier (UMI)). In some embodiments,the unique molecular identifying sequence can include from about 5 toabout 8 nucleotides. Alternatively, the unique molecular identifyingsequence can include less than about 5 or more than about 8 nucleotides.The unique molecular identifying sequence can be a unique sequence thatvaries across individual nucleic acid molecules coupled to a singlebead.

In some embodiments, the unique molecular identifying sequence can be arandom sequence (e.g., such as a random N-mer sequence). For example,the UMI can provide a unique identifier of the starting mRNA moleculethat was captured, in order to allow quantitation of the number oforiginal expressed RNA.

In general, an individual bead can be coupled to any number ofindividual nucleic acid molecules, for example, from one to tens tohundreds of thousands or even millions of individual nucleic acidmolecules. The respective barcodes for the individual nucleic acidmolecules can include both common sequence segments or relatively commonsequence segments and variable or unique sequence segments betweendifferent individual nucleic acid molecules coupled to the same bead.

Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules can include a common barcode sequence segment.However, the transcripts made from the different mRNA molecules within agiven partition can vary at the unique molecular identifying sequencesegment (e.g., UMI segment). Beneficially, even following any subsequentamplification of the contents of a given partition, the number ofdifferent UMIs can be indicative of the quantity of mRNA originatingfrom a given partition. As noted above, the transcripts can beamplified, cleaned up, and sequenced to identify the sequence of thecDNA transcript of the mRNA, as well as to sequence the barcode segmentand the UMI segment. While a poly(T) primer sequence is described, othertargeted or random priming sequences can also be used in priming thereverse transcription reaction. Likewise, although described asreleasing the barcoded oligonucleotides into the partition, in somecases, the nucleic acid molecules bound to the bead can be used tohybridize and capture the mRNA on the solid phase of the bead, forexample, in order to facilitate the separation of the RNA from othercell contents.

In some embodiments, precursors that include a functional group that isreactive or capable of being activated such that it becomes reactive canbe polymerized with other precursors to generate gel beads that includethe activated or activatable functional group. The functional group canthen be used to attach additional species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) to the gel beads. For example,some precursors featuring a carboxylic acid (COOH) group canco-polymerize with other precursors to form a bead that also includes aCOOH functional group. In some cases, acrylic acid (a species comprisingfree COOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a bead with free COOH groups. TheCOOH groups of the bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

In some embodiments, a degradable bead can be introduced into apartition, such that the bead degrades within the partition and anyassociated species (e.g., oligonucleotides) are released within thedroplet when the appropriate stimulus is applied. The free species(e.g., oligonucleotides, nucleic acid molecules) can interact with otherreagents contained in the partition. For example, a polyacrylamide beadfeaturing cystamine and linked, via a disulfide bond, to a barcodesequence, can be combined with a reducing agent within a droplet of awater-in-oil emulsion. Within the droplet, the reducing agent can breakthe various disulfide bonds, resulting in bead degradation and releaseof the barcode sequence into the aqueous, inner environment of thedroplet. In another example, heating of a droplet with a bead-boundbarcode sequence in basic solution can also result in bead degradationand release of the attached barcode sequence into the aqueous, innerenvironment of the droplet.

Any suitable number of species (e.g., primer, barcoded oligonucleotide)can be associated with a bead such that, upon release from the bead, thespecies (e.g., primer, e.g., barcoded oligonucleotide) are present inthe partition at a pre-defined concentration. Such pre-definedconcentration can be selected to facilitate certain reactions forgenerating a sequencing library, e.g., amplification, within thepartition. In some cases, the pre-defined concentration of the primercan be limited by the process of producing nucleic acid molecule (e.g.,oligonucleotide) bearing beads.

A degradable bead can include one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimulus,the bond is broken and the bead degrades. The labile bond can be achemical bond (e.g., covalent bond, ionic bond) or can be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some embodiments, a cross-linker used togenerate a bead can include a labile bond. Upon exposure to theappropriate conditions, the labile bond can be broken and the beaddegraded. For example, upon exposure of a polyacrylamide gel bead thatincludes cystamine cross-linkers to a reducing agent, the disulfidebonds of the cystamine can be broken and the bead degraded.

A degradable bead can be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc.) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species can have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some embodiments, a species can also be attached to a degradable beadvia a degradable linker (e.g., disulfide linker). The degradable linkercan respond to the same stimuli as the degradable bead or the twodegradable species can respond to different stimuli. For example, abarcode sequence can be attached, via a disulfide bond, to apolyacrylamide bead comprising cystamine. Upon exposure of thebarcoded-bead to a reducing agent, the bead degrades and the barcodesequence is released upon breakage of both the disulfide linkage betweenthe barcode sequence and the bead and the disulfide linkages of thecystamine in the bead.

As will be appreciated from the above description, while referred to asdegradation of a bead, in many embodiments, degradation can refer to thedisassociation of a bound or entrained species from a bead, both withand without structurally degrading the physical bead itself. Forexample, entrained species can be released from beads through osmoticpressure differences due to, for example, changing chemicalenvironments. By way of example, alteration of bead pore volumes due toosmotic pressure differences can generally occur without structuraldegradation of the bead itself. In some cases, an increase in porevolume due to osmotic swelling of a bead can permit the release ofentrained species within the bead. In some embodiments, osmoticshrinking of a bead can cause a bead to better retain an entrainedspecies due to pore volume contraction.

Numerous chemical triggers can be used to trigger the degradation ofbeads within partitions. Examples of these chemical changes can include,but are not limited to pH-mediated changes to the integrity of acomponent within the bead, degradation of a component of a bead viacleavage of cross-linked bonds, and depolymerization of a component of abead.

In some embodiments, a bead can be formed from materials that includedegradable chemical cross-linkers, such as BAC or cystamine. Degradationof such degradable cross-linkers can be accomplished through a number ofmechanisms. In some examples, a bead can be contacted with a chemicaldegrading agent that can induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent can be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent can degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead.

In certain embodiments, a change in pH of a solution, such as anincrease in pH, can trigger degradation of a bead. In other embodiments,exposure to an aqueous solution, such as water, can trigger hydrolyticdegradation, and thus degradation of the bead. In some cases, anycombination of stimuli can trigger degradation of a bead. For example, achange in pH can enable a chemical agent (e.g., DTT) to become aneffective reducing agent.

Beads can also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat can cause melting of a bead such that a portion of thebead degrades. In other cases, heat can increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat canalso act upon heat-sensitive polymers used as materials to constructbeads.

In addition to beads and analytes, partitions that are formed caninclude a variety of different reagents and species. For example, whenlysis reagents are present within the partitions, the lysis reagents canfacilitate the release of analytes within the partition. Examples oflysis agents include bioactive reagents, such as lysis enzymes that areused for lysis of different cell types, e.g., gram positive or negativebacteria, plants, yeast, mammalian, etc., such as lysozymes,achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and avariety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc.(St. Louis, Mo.), as well as other commercially available lysis enzymes.Other lysis agents can additionally or alternatively be co-partitionedto cause the release analytes into the partitions. For example, in somecases, surfactant-based lysis solutions can be used to lyse cells,although these can be less desirable for emulsion based systems wherethe surfactants can interfere with stable emulsions. In someembodiments, lysis solutions can include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some embodiments, lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic ormechanical cellular disruption can also be used in certain embodiments,e.g., non-emulsion based partitioning such as encapsulation of analytesthat can be in addition to or in place of droplet partitioning, whereany pore volume of the encapsulate is sufficiently small to retainnucleic acid fragments of a given size, following cellular disruption.

Examples of other species that can be co-partitioned with analytes inthe partitions include, but are not limited to, DNase and RNaseinactivating agents or inhibitors or chelating agents, such as EDTA, andother reagents employed in removing or otherwise reducing negativeactivity or impact of different cell lysate components on subsequentprocessing of nucleic acids. Additional reagents can also beco-partitioned, including endonucleases to fragment DNA, DNA polymeraseenzymes and dNTPs used to amplify nucleic acid fragments and to attachthe barcode molecular tags to the amplified fragments.

Additional reagents can also include reverse transcriptase enzymes,including enzymes with terminal transferase activity, primers andoligonucleotides, and switch oligonucleotides (also referred to hereinas “switch oligos” or “template switching oligonucleotides”) which canbe used for template switching. In some embodiments, template switchingcan be used to increase the length of a cDNA. Template switching can beused to append a predefined nucleic acid sequence to the cDNA. In anexample of template switching, cDNA can be generated from reversetranscription of a template, e.g., cellular mRNA, where a reversetranscriptase with terminal transferase activity can add additionalnucleotides, e.g., poly(C), to the cDNA in a template independentmanner. Switch oligos can include sequences complementary to theadditional nucleotides, e.g., poly(G). The additional nucleotides (e.g.,poly(C)) on the cDNA can hybridize to the additional nucleotides (e.g.,poly(G)) on the switch oligo, whereby the switch oligo can be used bythe reverse transcriptase as template to further extend the cDNA.

Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target. In some cases, the hybridizationregion includes a series of G bases to complement the overhanging Cbases at the 3′ end of a cDNA molecule. The series of G bases caninclude 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or morethan 5 G bases. The template sequence can include any sequence to beincorporated into the cDNA. In some cases, the template region includesat least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/orfunctional sequences. Switch oligos can include deoxyribonucleic acids;ribonucleic acids; modified nucleic acids including 2-Aminopurine,2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC,2′-deoxylnosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleicacids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), andcombinations of the foregoing.

In some embodiments, beads that are partitioned with the analyte caninclude different types of oligonucleotides bound to the bead, where thedifferent types of oligonucleotides bind to different types of analytes.For example, a bead can include one or more first oligonucleotides(which can be capture probes, for example) that can bind or hybridize toa first type of analyte, such as mRNA for example, and one or moresecond oligonucleotides (which can be capture probes, for example) thatcan bind or hybridize to a second type of analyte, such as gDNA forexample. Partitions can also include lysis agents that aid in releasingnucleic acids from the co-partitioned cell, and can also include anagent (e.g., a reducing agent) that can degrade the bead and/or breakcovalent linkages between the oligonucleotides and the bead, releasingthe oligonucleotides into the partition. The released barcodedoligonucleotides (which can also be barcoded) can hybridize with mRNAreleased from the cell and also with gDNA released from the cell.

Barcoded constructs thus formed from hybridization can include a firsttype of construct that includes a sequence corresponding to an originalbarcode sequence from the bead and a sequence corresponding to atranscript from the cell, and a second type of construct that includes asequence corresponding to the original barcode sequence from the beadand a sequence corresponding to genomic DNA from the cell. The barcodedconstructs can then be released/removed from the partition and, in someembodiments, further processed to add any additional sequences. Theresulting constructs can then be sequenced, the sequencing dataprocessed, and the results used to spatially characterize the mRNA andthe gDNA from the cell.

In another example, a partition includes a bead that includes a firsttype of oligonucleotide (e.g., a first capture probe) with a firstbarcode sequence, a poly(T) priming sequence that can hybridize with thepoly(A) tail of an mRNA transcript, and a UMI barcode sequence that canuniquely identify a given transcript. The bead also includes a secondtype of oligonucleotide (e.g., a second capture probe) with a secondbarcode sequence, a targeted priming sequence that is capable ofspecifically hybridizing with a third barcoded oligonucleotide (e.g., ananalyte capture agent) coupled to an antibody that is bound to thesurface of the partitioned cell. The third barcoded oligonucleotideincludes a UMI barcode sequence that uniquely identifies the antibody(and thus, the particular cell surface feature to which it is bound).

In this example, the first and second barcoded oligonucleotides includethe same spatial barcode sequence (e.g., the first and second barcodesequences are the same), which permits downstream association ofbarcoded nucleic acids with the partition. In some embodiments, however,the first and second barcode sequences are different.

The partition also includes lysis agents that aid in releasing nucleicacids from the cell and can also include an agent (e.g., a reducingagent) that can degrade the bead and/or break a covalent linkage betweenthe barcoded oligonucleotides and the bead, releasing them into thepartition. The first type of released barcoded oligonucleotide canhybridize with mRNA released from the cell and the second type ofreleased barcoded oligonucleotide can hybridize with the third type ofbarcoded oligonucleotide, forming barcoded constructs.

The first type of barcoded construct includes a spatial barcode sequencecorresponding to the first barcode sequence from the bead and a sequencecorresponding to the UMI barcode sequence from the first type ofoligonucleotide, which identifies cell transcripts. The second type ofbarcoded construct includes a spatial barcode sequence corresponding tothe second barcode sequence from the second type of oligonucleotide, anda UMI barcode sequence corresponding to the third type ofoligonucleotide (e.g., the analyte capture agent) and used to identifythe cell surface feature. The barcoded constructs can then bereleased/removed from the partition and, in some embodiments, furtherprocessed to add any additional sequences. The resulting constructs arethen sequenced, sequencing data processed, and the results used tocharacterize the mRNA and cell surface feature of the cell.

The foregoing discussion involves two specific examples of beads witholigonucleotides for analyzing two different analytes within apartition. More generally, beads that are partitioned can have any ofthe structures described previously, and can include any of thedescribed combinations of oligonucleotides for analysis of two or more(e.g., three or more, four or more, five or more, six or more, eight ormore, ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 ormore, 40 or more, 50 or more) different types of analytes within apartition. Examples of beads with combinations of different types ofoligonucleotides (e.g., capture probes) for concurrently analyzingdifferent combinations of analytes within partitions include, but arenot limited to: (a) genomic DNA and cell surface features (e.g., usingthe analyte capture agents described herein); (b) mRNA and a lineagetracing construct; (c) mRNA and cell methylation status; (d) mRNA andaccessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq); (e)mRNA and cell surface or intracellular proteins and/or metabolites; (f)a barcoded analyte capture agent (e.g., the MHC multimers describedherein) and a V(D)J sequence of an immune cell receptor (e.g., T-cellreceptor); and (g) mRNA and a perturbation agent (e.g., a CRISPRcrRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein). In some embodiments, aperturbation agent can be a small molecule, an antibody, a drug, anaptamer, a nucleic acid (e.g., miRNA), a physical environmental (e.g.,temperature change), or any other known perturbation agents.

(e) Sequencing Analysis

After analytes from the sample have hybridized or otherwise beenassociated with capture probes, analyte capture agents, or otherbarcoded oligonucleotide sequences according to any of the methodsdescribed above in connection with the general spatial cell-basedanalytical methodology, the barcoded constructs that result fromhybridization/association are analyzed via sequencing to identify theanalytes.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample. Alternatively, if the barcoded sample has been separated intofragments, cell groups, or individual cells, as described above,sequencing can be performed on individual fragments, cell groups, orcells. For analytes that have been barcoded via partitioning with beads,as described above, individual analytes (e.g., cells, or cellularcontents following lysis of cells) can be extracted from the partitionsby breaking the partitions, and then analyzed by sequencing to identifythe analytes.

A wide variety of different sequencing methods can be used to analyzebarcoded analyte constructs. In general, sequenced polynucleotides canbe, for example, nucleic acid molecules such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA), including variants or derivativesthereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acidmolecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various commercialsystems. More generally, sequencing can be performed using nucleic acidamplification, polymerase chain reaction (PCR) (e.g., digital PCR anddroplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplexPCR, PCR-based singleplex methods, emulsion PCR), and/or isothermalamplification.

Other examples of methods for sequencing genetic material include, butare not limited to, DNA hybridization methods (e.g., Southern blotting),restriction enzyme digestion methods, Sanger sequencing methods,next-generation sequencing methods (e.g., single-molecule real-timesequencing, nanopore sequencing, and Polony sequencing), ligationmethods, and microarray methods. Additional examples of sequencingmethods that can be used include targeted sequencing, single moleculereal-time sequencing, exon sequencing, electron microscopy-basedsequencing, panel sequencing, transistor-mediated sequencing, directsequencing, random shotgun sequencing, Sanger dideoxy terminationsequencing, whole-genome sequencing, sequencing by hybridization,pyrosequencing, capillary electrophoresis, gel electrophoresis, duplexsequencing, cycle sequencing, single-base extension sequencing,solid-phase sequencing, high-throughput sequencing, massively parallelsignature sequencing, co-amplification at lower denaturationtemperature-PCR (COLD-PCR), sequencing by reversible dye terminator,paired-end sequencing, near-term sequencing, exonuclease sequencing,sequencing by ligation, short-read sequencing, single-moleculesequencing, sequencing-by-synthesis, real-time sequencing,reverse-terminator sequencing, nanopore sequencing, MS-PET sequencing,and any combinations thereof.

Sequence analysis of the nucleic acid molecules (including barcodednucleic acid molecules or derivatives thereof) can be direct orindirect. Thus, the sequence analysis substrate (which can be viewed asthe molecule which is subjected to the sequence analysis step orprocess) can directly be the barcoded nucleic acid molecule or it can bea molecule which is derived therefrom (e.g., a complement thereof).Thus, for example, in the sequence analysis step of a sequencingreaction, the sequencing template can be the barcoded nucleic acidmolecule or it can be a molecule derived therefrom. For example, a firstand/or second strand DNA molecule can be directly subjected to sequenceanalysis (e.g., sequencing), i.e., can directly take part in thesequence analysis reaction or process (e.g., the sequencing reaction orsequencing process, or be the molecule which is sequenced or otherwiseidentified). Alternatively, the barcoded nucleic acid molecule can besubjected to a step of second strand synthesis or amplification beforesequence analysis (e.g., sequencing or identification by anothertechnique). The sequence analysis substrate (e.g., template) can thus bean amplicon or a second strand of a barcoded nucleic acid molecule.

In some embodiments, both strands of a double stranded molecule can besubjected to sequence analysis (e.g., sequenced). In some embodiments,single stranded molecules (e.g., barcoded nucleic acid molecules) can beanalyzed (e.g., sequenced). To perform single molecule sequencing, thenucleic acid strand can be modified at the 3′ end.

In some embodiments, massively parallel pyrosequencing techniques can beused for sequencing nucleic acids. In pyrosequencing, the nucleic acidis amplified inside water droplets in an oil solution (emulsion PCR),with each droplet containing a single nucleic acid template attached toa single primer-coated bead that then forms a clonal colony. Thesequencing system contains many picolitre-volume wells each containing asingle bead and sequencing enzymes. Pyrosequencing uses luciferase togenerate light for detection of the individual nucleotides added to thenascent nucleic acid and the combined data are used to generate sequencereads.

As another example application of pyrosequencing, released PPi can bedetected by being immediately converted to adenosine triphosphate (ATP)by ATP sulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons, such as described in Ronaghi, et al., Anal.Biochem. 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001);Ronaghi et al. Science 281 (5375), 363 (1998); and U.S. Pat. Nos.6,210,891, 6,258,568, and 6,274,320, the entire contents of each ofwhich are incorporated herein by reference.

Massively parallel sequencing techniques can be used for sequencingnucleic acids, as described above. In one embodiment, a massivelyparallel sequencing technique can be based on reversibledye-terminators. As an example, DNA molecules are first attached toprimers on, e.g., a glass or silicon substrate, and amplified so thatlocal clonal colonies are formed (e.g., by bridge amplification). Fourtypes of ddNTPs are added, and non-incorporated nucleotides are washedaway. Unlike pyrosequencing, the DNA is only extended one nucleotide ata time due to a blocking group (e.g., 3′ blocking group present on thesugar moiety of the ddNTP). A detector acquires images of thefluorescently labelled nucleotides, and then the dye along with theterminal 3′ blocking group is chemically removed from the DNA, as aprecursor to a subsequent cycle. This process can be repeated until therequired sequence data is obtained.

In some embodiments, sequencing is performed by detection of hydrogenions that are released during the polymerization of DNA. A microwellcontaining a template DNA strand to be sequenced can be flooded with asingle type of nucleotide. If the introduced nucleotide is complementaryto the leading template nucleotide, it is incorporated into the growingcomplementary strand. This causes the release of a hydrogen ion thattriggers a hypersensitive ion sensor, which indicates that a reactionhas occurred. If homopolymer repeats are present in the templatesequence, multiple nucleotides will be incorporated in a single cycle.This leads to a corresponding number of released hydrogen ions and aproportionally higher electronic signal.

In some embodiments, sequencing can be performed in situ. In situsequencing methods are particularly useful, for example, when thebiological sample remains intact after analytes on the sample surface(e.g., cell surface analytes) or within the sample (e.g., intracellularanalytes) have been barcoded. In situ sequencing typically involvesincorporation of a labeled nucleotide (e.g., fluorescently labeledmononucleotides or dinucleotides) in a sequential, template-dependentmanner or hybridization of a labeled primer (e.g., a labeled randomhexamer) to a nucleic acid template such that the identities (i.e.,nucleotide sequence) of the incorporated nucleotides or labeled primerextension products can be determined, and consequently, the nucleotidesequence of the corresponding template nucleic acid. Aspects of in situsequencing are described, for example, in Mitra et al., (2003) Anal.Biochem. 320, 55-65, and Lee et al., (2014) Science, 343(6177),1360-1363, the entire contents of each of which are incorporated hereinby reference.

In addition, examples of methods and systems for performing in situsequencing are described in PCT Patent Application Publication Nos.WO2014/163886, WO2018/045181, WO2018/045186, and in U.S. Pat. Nos.10,138,509 and 10,179,932, the entire contents of each of which areincorporated herein by reference. Example techniques for in situsequencing include, but are not limited to, STARmap (described forexample in Wang et al., (2018) Science, 361(6499) 5691), MERFISH(described for example in Moffitt, (2016) Methods in Enzymology, 572,1-49), and FISSEQ (described for example in U.S. Patent ApplicationPublication No. 2019/0032121). The entire contents of each of theforegoing references are incorporated herein by reference.

For analytes that have been barcoded via partitioning, barcoded nucleicacid molecules or derivatives thereof (e.g., barcoded nucleic acidmolecules to which one or more functional sequences have been added, orfrom which one or more features have been removed) can be pooled andprocessed together for subsequent analysis such as sequencing on highthroughput sequencers. Processing with pooling can be implemented usingbarcode sequences. For example, barcoded nucleic acid molecules of agiven partition can have the same barcode, which is different frombarcodes of other spatial partitions. Alternatively, barcoded nucleicacid molecules of different partitions can be processed separately forsubsequent analysis (e.g., sequencing).

In some embodiments, where capture probes do not contain a spatialbarcode, the spatial barcode can be added after the capture probecaptures analytes from a biological sample and before analysis of theanalytes. When a spatial barcode is added after an analyte is captured,the barcode can be added after amplification of the analyte (e.g.,reverse transcription and polymerase amplification of RNA). In someembodiments, analyte analysis uses direct sequencing of one or morecaptured analytes, such as direct sequencing of hybridized RNA. In someembodiments, direct sequencing is performed after reverse transcriptionof hybridized RNA. In some embodiments direct sequencing is performedafter amplification of reverse transcription of hybridized RNA.

In some embodiments, direct sequencing of captured RNA is performed bysequencing-by-synthesis (SBS). In some embodiments, a sequencing primeris complementary to a sequence in one or more of the domains of acapture probe (e.g., functional domain). In such embodiments,sequencing-by-synthesis can include reverse transcription and/oramplification in order to generate a template sequence (e.g., functionaldomain) from which a primer sequence can bind.

SBS can involve hybridizing an appropriate primer, sometimes referred toas a sequencing primer, with the nucleic acid template to be sequenced,extending the primer, and detecting the nucleotides used to extend theprimer. Preferably, the nucleic acid used to extend the primer isdetected before a further nucleotide is added to the growing nucleicacid chain, thus allowing base-by-base in situ nucleic acid sequencing.The detection of incorporated nucleotides is facilitated by includingone or more labelled nucleotides in the primer extension reaction. Toallow the hybridization of an appropriate sequencing primer to thenucleic acid template to be sequenced, the nucleic acid template shouldnormally be in a single stranded form. If the nucleic acid templatesmaking up the nucleic acid features are present in a double strandedform these can be processed to provide single stranded nucleic acidtemplates using methods well known in the art, for example bydenaturation, cleavage, etc. The sequencing primers which are hybridizedto the nucleic acid template and used for primer extension arepreferably short oligonucleotides, for example, 15 to 25 nucleotides inlength. The sequencing primers can be greater than 25 nucleotides inlength as well. For example, sequencing primers can be about 20 to about60 nucleotides in length, or more than 60 nucleotides in length. Thesequencing primers can be provided in solution or in an immobilizedform. Once the sequencing primer has been annealed to the nucleic acidtemplate to be sequenced by subjecting the nucleic acid template andsequencing primer to appropriate conditions, primer extension is carriedout, for example using a nucleic acid polymerase and a supply ofnucleotides, at least some of which are provided in a labelled form, andconditions suitable for primer extension if a suitable nucleotide isprovided.

Preferably after each primer extension step, a washing step is includedin order to remove unincorporated nucleotides which can interfere withsubsequent steps. Once the primer extension step has been carried out,the nucleic acid colony is monitored to determine whether a labellednucleotide has been incorporated into an extended primer. The primerextension step can then be repeated to determine the next and subsequentnucleotides incorporated into an extended primer. If the sequence beingdetermined is unknown, the nucleotides applied to a given colony areusually applied in a chosen order which is then repeated throughout theanalysis, for example dATP, dTTP, dCTP, dGTP.

SBS techniques which can be used are described for example, but notlimited to, those in U.S. Patent App. Pub. No. 2007/0166705, U.S. PatentApp. Pub. No. 2006/0188901, U.S. Pat. No. 7,057,026, U.S. Patent App.Pub. No. 2006/0240439, U.S. Patent App. Pub. No. 2006/0281109, PCTPatent App. Pub. No. WO 05/065814, U.S. Patent App. Pub. No.2005/0100900, PCT Patent App. Pub. No. WO 06/064199, PCT Patent App.Pub. No. WO07/010,251, U.S. Patent App. Pub. No. 2012/0270305, U.S.Patent App. Pub. No. 2013/0260372, and U.S. Patent App. Pub. No.2013/0079232, the entire contents of each of which are incorporatedherein by reference.

In some embodiments, direct sequencing of captured RNA is performed bysequential fluorescence hybridization (e.g., sequencing byhybridization). In some embodiments, a hybridization reaction where RNAis hybridized to a capture probe is performed in situ. In someembodiments, captured RNA is not amplified prior to hybridization with asequencing probe. In some embodiments, RNA is amplified prior tohybridization with sequencing probes (e.g., reverse transcription tocDNA and amplification of cDNA). In some embodiments, amplification isperformed using single-molecule hybridization chain reaction. In someembodiments, amplification is performed using rolling chainamplification.

Sequential fluorescence hybridization can involve sequentialhybridization of probes including degenerate primer sequences and adetectable label. A degenerate primer sequence is a shortoligonucleotide sequence which is capable of hybridizing to any nucleicacid fragment independent of the sequence of said nucleic acid fragment.For example, such a method could include the steps of: (a) providing amixture including four probes, each of which includes either A, C, G, orT at the 5′-terminus, further including degenerate nucleotide sequenceof 5 to 11 nucleotides in length, and further including a functionaldomain (e.g., fluorescent molecule) that is distinct for probes with A,C, G, or T at the 5′-terminus; (b) associating the probes of step (a) tothe target polynucleotide sequences, whose sequence needs will bedetermined by this method; (c) measuring the activities of the fourfunctional domains and recording the relative spatial location of theactivities; (d) removing the reagents from steps (a)-(b) from the targetpolynucleotide sequences; and repeating steps (a)-(d) for n cycles,until the nucleotide sequence of the spatial domain for each bead isdetermined, with modification that the oligonucleotides used in step (a)are complementary to part of the target polynucleotide sequences and thepositions 1 through n flanking the part of the sequences. Because thebarcode sequences are different, in some embodiments, these additionalflanking sequences are degenerate sequences. The fluorescent signal fromeach spot on the array for cycles 1 through n can be used to determinethe sequence of the target polynucleotide sequences.

In some embodiments, direct sequencing of captured RNA using sequentialfluorescence hybridization is performed in vitro. In some embodiments,captured RNA is amplified prior to hybridization with a sequencing probe(e.g., reverse transcription to cDNA and amplification of cDNA). In someembodiments, a capture probe containing captured RNA is exposed to thesequencing probe targeting coding regions of RNA. In some embodiments,one or more sequencing probes are targeted to each coding region. Insome embodiments, the sequencing probe is designed to hybridize withsequencing reagents (e.g., a dye-labeled readout oligonucleotides). Asequencing probe can then hybridize with sequencing reagents. In someembodiments, output from the sequencing reaction is imaged. In someembodiments, a specific sequence of cDNA is resolved from an image of asequencing reaction. In some embodiments, reverse transcription ofcaptured RNA is performed prior to hybridization to the sequencingprobe. In some embodiments, the sequencing probe is designed to targetcomplementary sequences of the coding regions of RNA (e.g., targetingcDNA).

In some embodiments, a captured RNA is directly sequenced using ananopore-based method. In some embodiments, direct sequencing isperformed using nanopore direct RNA sequencing in which captured RNA istranslocated through a nanopore. A nanopore current can be recorded andconverted into a base sequence. In some embodiments, captured RNAremains attached to a substrate during nanopore sequencing. In someembodiments, captured RNA is released from the substrate prior tonanopore sequencing. In some embodiments, where the analyte of interestis a protein, direct sequencing of the protein can be performed usingnanopore-based methods. Examples of nanopore-based sequencing methodsthat can be used are described in Deamer et al., Trends Biotechnol. 18,14 7-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li etal., Nat. Mater. 2:611-615 (2003); Soni et al., Clin. Chem. 53,1996-2001 (2007); Healy et al., Nanomed. 2, 459-481 (2007); Cockroft etal., J. Am. Chem. Soc. 130, 818-820 (2008); and in U.S. Pat. No.7,001,792. The entire contents of each of the foregoing references areincorporated herein by reference.

In some embodiments, direct sequencing of captured RNA is performedusing single molecule sequencing by ligation. Such techniques utilizeDNA ligase to incorporate oligonucleotides and identify theincorporation of such oligonucleotides. The oligonucleotides typicallyhave different labels that are correlated with the identity of aparticular nucleotide in a sequence to which the oligonucleotideshybridize. Aspects and features involved in sequencing by ligation aredescribed, for example, in Shendure et al. Science (2005), 309:1728-1732, and in U.S. Pat. Nos. 5,599,675; 5,750,341; 6,969,488;6,172,218; and 6,306,597, the entire contents of each of which areincorporated herein by reference.

In some embodiments, nucleic acid hybridization can be used forsequencing. These methods utilize labeled nucleic acid decoder probesthat are complementary to at least a portion of a barcode sequence.Multiplex decoding can be performed with pools of many different probeswith distinguishable labels. Non-limiting examples of nucleic acidhybridization sequencing are described for example in U.S. Pat. No.8,460,865, and in Gunderson et al., Genome Research 14:870-877 (2004),the entire contents of each of which are incorporated herein byreference.

In some embodiments, commercial high-throughput digital sequencingtechniques can be used to analyze barcode sequences, in which DNAtemplates are prepared for sequencing not one at a time, but in a bulkprocess, and where many sequences are read out preferably in parallel,or alternatively using an ultra-high throughput serial process thatitself may be parallelized. Examples of such techniques includeIllumina® sequencing (e.g., flow cell-based sequencing techniques),sequencing by synthesis using modified nucleotides (such ascommercialized in TruSeq™ and HiSeq™ technology by Illumina, Inc., SanDiego, Calif.), HeliScope™ by Helicos Biosciences Corporation,Cambridge, Mass., and PacBio RS by Pacific Biosciences of California,Inc., Menlo Park, Calif.), sequencing by ion detection technologies (IonTorrent, Inc., South San Francisco, Calif.), and sequencing of DNAnanoballs (Complete Genomics, Inc., Mountain View, Calif.).

In some embodiments, detection of a proton released upon incorporationof a nucleotide into an extension product can be used in the methodsdescribed herein. For example, the sequencing methods and systemsdescribed in U.S. Patent Application Publication Nos. 2009/0026082,2009/0127589, 2010/0137143, and 2010/0282617, can be used to directlysequence barcodes. The entire contents of each of the foregoingreferences are incorporated herein by reference.

In some embodiments, real-time monitoring of DNA polymerase activity canbe used during sequencing. For example, nucleotide incorporations can bedetected through fluorescence resonance energy transfer (FRET), asdescribed for example in Levene et al., Science (2003), 299, 682-686,Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al.,Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181. The entire contentsof each of the foregoing references are herein incorporated byreference.

IV. Multiplexing

(a) Multiplexing Generally

In various embodiments of spatial analysis as described herein, featurescan include different types of capture probes for analyzing bothintrinsic and extrinsic information for individual cells. For example, afeature can include one or more of the following: 1) a capture probefeaturing a capture domain that binds to one or more endogenous nucleicacids in the cell; 2) a capture probe featuring a capture domain thatbinds to one or more exogenous nucleic acids in the cell (e.g., nucleicacids from a microorganism (e.g., a virus, a bacterium)) that infectsthe cell, nucleic acids introduced into the cell (e.g., such as plasmidsor nucleic acid derived therefrom), nucleic acids for gene editing(e.g., CRISPR-related RNA such as crRNA, guide RNA); 3) a capture probefeaturing a capture domain that binds to an analyte capture agent (e.g.,an antibody coupled to a oligonucleotide that includes a capture agentbarcode domain having an analyte capture sequence that binds the capturedomain), and 4) a capture moiety featuring a domain that binds to aprotein (e.g., an exogenous protein expressed in the cell, a proteinfrom a microorganism (e.g., a virus, a bacterium)) that infects thecell, or a binding partner for a protein of the cell (e.g., an antigenfor an immune cell receptor).

In some embodiments of any of the spatial analysis methods as describedherein, spatial profiling includes concurrent analysis of two differenttypes of analytes. A feature can be a gel bead, which is coupled (e.g.,reversibly coupled) to one or more capture probes. The capture probescan include a spatial barcode sequence and a poly(T) priming sequencethat can hybridize with the poly(A) tail of an mRNA transcript. Thecapture probe can also include a UMI sequence that can uniquely identifya given transcript. The capture probe can also include a spatial barcodesequence and a random N-mer priming sequence that is capable of randomlyhybridizing with gDNA. In this configuration, capture probes can includethe same spatial barcode sequence, which permits association ofdownstream sequencing reads with the feature.

In some embodiments of any of the spatial analysis methods as describedherein, a feature can be a gel bead, which is coupled (e.g., reversiblycoupled) to capture probes. The Capture probe can include a spatialbarcode sequence and a poly(T) priming sequence that can hybridize withthe poly(A) tail of an mRNA transcript. The capture probe can alsoinclude a UMI sequence that can uniquely identify a given transcript.The capture probe can include a spatial barcode sequence and a capturedomain that is capable of specifically hybridizing with an analytecapture agent. The analyte capture agent can includes an oligonucleotidethat includes an analyte capture sequence that interacts with thecapture domain coupled to the feature. The oligonucleotide of theanalyte capture agent can be coupled to an antibody that is bound to thesurface of a cell. The oligonucleotide includes a barcode sequence(e.g., an analyte binding moiety barcode) that uniquely identifies theantibody (and thus, the particular cell surface feature to which it isbound). In this configuration, the capture probes include the samespatial barcode sequence, which permit downstream association ofbarcoded nucleic acids with the location on the spatial array. In someembodiments of any of the spatial profiling methods described herein,the analyte capture agents can be can be produced by any suitable route,including via example coupling schemes described elsewhere herein.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of two or more biological analytes that canbe concurrently measured include, without limitation: (a) genomic DNAand cell surface features (e.g., via analyte capture agents that bind toa cell surface feature), (b) mRNA and a lineage tracing construct, (c)mRNA and cell methylation status, (d) mRNA and accessible chromatin(e.g., ATAC-seq, DNase-seq, and/or MNase-seq), (e) mRNA and cell surfaceor intracellular proteins and/or metabolites, (f) mRNA and chromatin(spatial organization of chromatin in a cell), (g) an analyte captureagent (e.g., any of the MHC multimers described herein) and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), (h) mRNAand a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein), (i)genomic DNA and a perturbation agent, (j) an analyte capture agent and aperturbation agents, (k) accessible chromatin and a perturbation agent,(l) chromatin (e.g., spatial organization of chromatin in a cell) and aperturbation agent, and (m) cell surface or intracellular proteinsand/or metabolites and a perturbation agent (e.g., any of theperturbation agents described herein), or any combination thereof.

In some embodiments of any of the spatial analysis methods describedherein, the first analyte can include a nucleic acid molecule with anucleic acid sequence (e.g., mRNA, complementary DNA derived fromreverse transcription of mRNA) encoding at least a portion of a V(D)Jsequence of an immune cell receptor (e.g., a TCR or BCR). In someembodiments, the nucleic acid molecule with a nucleic acid sequenceencoding at least a portion of a V(D)J sequence of an immune cellreceptor is cDNA first generated from reverse transcription of thecorresponding mRNA, using a poly(T) containing primer. The cDNA that isgenerated can then be barcoded using a primer, featuring a spatialbarcode sequence (and optionally, a UMI sequence) that hybridizes withat least a portion of the cDNA that is generated. In some embodiments, atemplate switching oligonucleotide in conjunction a terminal transferaseor a reverse transcriptase having terminal transferase activity can beemployed to generate a priming region on the cDNA to which a barcodedprimer can hybridize during cDNA generation. Terminal transferaseactivity can, for example, add a poly(C) tail to a 3′ end of the cDNAsuch that the template switching oligonucleotide can bind via a poly(G)priming sequence and the 3′ end of the cDNA can be further extended. Theoriginal mRNA template and template switching oligonucleotide can thenbe denatured from the cDNA and the barcoded primer comprising a sequencecomplementary to at least a portion of the generated priming region onthe cDNA can then hybridize with the cDNA and a barcoded constructcomprising the barcode sequence (and any optional UMI sequence) and acomplement of the cDNA generated. Additional methods and compositionssuitable for barcoding cDNA generated from mRNA transcripts includingthose encoding V(D)J regions of an immune cell receptor and/or barcodingmethods and composition including a template switch oligonucleotide aredescribed, for example, in PCT Patent Application Publication No. WO2018/075693, and in U.S. Patent Application Publication No.2018/0105808, the entire contents of each of which are incorporatedherein by reference.

In some embodiments, V(D)J analysis can be performed using methodssimilar to those described herein. For example, V(D)J analysis can becompleted with the use of one or more analyte capture agents that bindto particular surface features of immune cells and are associated withbarcode sequences (e.g., analyte binding moiety barcodes). The one ormore analyte capture agents can include an MHC or MHC multimer. Abarcoded oligonucleotide coupled to a bead that can be used for V(D)Janalysis. The oligonucleotide is coupled to a bead by a releasablelinkage, such as a disulfide linker. The oligonucleotide can includefunctional sequences that are useful for subsequent processing, such asfunctional sequence, which can include a sequencer specific flow cellattachment sequence, e.g., a P5 sequence, as well as functionalsequence, which can include sequencing primer sequences, e.g., a R1primer binding site. In some embodiments, the sequence can include a P7sequence and a R2 primer binding site. A barcode sequence can beincluded within the structure for use in barcoding the templatepolynucleotide. The functional sequences can be selected forcompatibility with a variety of different sequencing systems, e.g., IonTorrent Proton or PGM, Illumina sequencing instruments, etc., and therequirements thereof. In some embodiments, the barcode sequence,functional sequences (e.g., flow cell attachment sequence) andadditional sequences (e.g., sequencing primer sequences) can be commonto all of the oligonucleotides attached to a given bead. The barcodedoligonucleotide can also include a sequence to facilitate templateswitching (e.g., a poly(G) sequence). In some embodiments, theadditional sequence provides a unique molecular identifier (UMI)sequence segment, as described elsewhere herein.

In an exemplary method of cellular polynucleotide analysis using abarcode oligonucleotide, a cell is co-partitioned along with a beadbearing a barcoded oligonucleotide and additional reagents such as areverse transcriptase, primers, oligonucleotides (e.g., templateswitching oligonucleotides), dNTPs, and a reducing agent into apartition (e.g., a droplet in an emulsion). Within the partition, thecell can be lysed to yield a plurality of template polynucleotides(e.g., DNA such as genomic DNA, RNA such as mRNA, etc.).

A reaction mixture featuring a template polynucleotide from a cell and(i) the primer having a sequence towards a 3′ end that hybridizes to thetemplate polynucleotide (e.g., poly(T)) and (ii) a template switchingoligonucleotide that includes a first oligonucleotide towards a 5′ endcan be subjected to an amplification reaction to yield a firstamplification product. In some embodiments, the template polynucleotideis an mRNA with a poly(A) tail and the primer that hybridizes to thetemplate polynucleotide includes a poly(T) sequence towards a 3′ end,which is complementary to the poly(A) segment. The first oligonucleotidecan include at least one of an adaptor sequence, a barcode sequence, aunique molecular identifier (UMI) sequence, a primer binding site, and asequencing primer binding site or any combination thereof. In somecases, a first oligonucleotide is a sequence that can be common to allpartitions of a plurality of partitions. For example, the firstoligonucleotide can include a flow cell attachment sequence, anamplification primer binding site, or a sequencing primer binding siteand the first amplification reaction facilitates the attachment theoligonucleotide to the template polynucleotide from the cell. In someembodiments, the first oligonucleotide includes a primer binding site.In some embodiments, the first oligonucleotide includes a sequencingprimer binding site.

The sequence towards a 3′ end (e.g., poly(T)) of the primer hybridizesto the template polynucleotide. In a first amplification reaction,extension reaction reagents, e.g., reverse transcriptase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence using the cell's nucleicacid as a template, to produce a transcript, e.g., cDNA, having afragment complementary to the strand of the cell's nucleic acid to whichthe primer annealed. In some embodiments, the reverse transcriptase hasterminal transferase activity and the reverse transcriptase addsadditional nucleotides, e.g., poly(C), to the cDNA in a templateindependent manner.

The template switching oligonucleotide, for example a template switchingoligonucleotide which includes a poly(G) sequence, can hybridize to thecDNA and facilitate template switching in the first amplificationreaction. The transcript, therefore, can include the sequence of theprimer, a sequence complementary to the template polynucleotide from thecell, and a sequence complementary to the template switchingoligonucleotide.

In some embodiments of any of the spatial analysis methods describedherein, subsequent to the first amplification reaction, the firstamplification product or transcript can be subjected to a secondamplification reaction to generate a second amplification product. Insome embodiments, additional sequences (e.g., functional sequences suchas flow cell attachment sequence, sequencing primer binding sequences,barcode sequences, etc.) are attached. The first and secondamplification reactions can be performed in the same volume, such as forexample in a droplet. In some embodiments, the first amplificationproduct is subjected to a second amplification reaction in the presenceof a barcoded oligonucleotide to generate a second amplification producthaving a barcode sequence. The barcode sequence can be unique to apartition, that is, each partition can have a unique barcode sequence.The barcoded oligonucleotide can include a sequence of at least asegment of the template switching oligonucleotide and at least a secondoligonucleotide. The segment of the template switching oligonucleotideon the barcoded oligonucleotide can facilitate hybridization of thebarcoded oligonucleotide to the transcript, e.g., cDNA, to facilitatethe generation of a second amplification product. In addition to abarcode sequence, the barcoded oligonucleotide can include a secondoligonucleotide such as at least one of an adaptor sequence, a uniquemolecular identifier (UMI) sequence, a primer binding site, and asequencing primer binding site, or any combination thereof.

In some embodiments of any of the spatial analysis methods describedherein, the second amplification reaction uses the first amplificationproduct as a template and the barcoded oligonucleotide as a primer. Insome embodiments, the segment of the template switching oligonucleotideon the barcoded oligonucleotide can hybridize to the portion of the cDNAor complementary fragment having a sequence complementary to thetemplate switching oligonucleotide or that which was copied from thetemplate switching oligonucleotide. In the second amplificationreaction, extension reaction reagents, e.g., polymerase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence using the firstamplification product as template. The second amplification product caninclude a second oligonucleotide, a sequence of a segment of thetemplate polynucleotide (e.g., mRNA), and a sequence complementary tothe primer.

In some embodiments of any of the spatial analysis methods describedherein, the second amplification product uses the barcodedoligonucleotide as a template and at least a portion of the firstamplification product as a primer. The segment of the firstamplification product (e.g., cDNA) having a sequence complementary tothe template switching oligonucleotide can hybridize to the segment ofthe barcoded oligonucleotide comprising a sequence of at least a segmentof the template switching oligonucleotide. In the second amplificationreaction, extension reaction reagents, e.g., polymerase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence (e.g., firstamplification product) using the barcoded oligonucleotide as template.The second amplification product can include the sequence of the primer,a sequence which is complementary to the sequence of the templatepolynucleotide (e.g., mRNA), and a sequence complementary to the secondoligonucleotide.

In some embodiments of any of the spatial analysis methods describedherein, three or more classes of biological analytes can be concurrentlymeasured. For example, a feature can include capture probes that canparticipate in an assay of at least three different types of analytesvia three different capture domains. A bead can be coupled to a barcodedoligonucleotide that includes a capture domain that includes a poly(T)priming sequence for mRNA analysis; a barcoded oligonucleotide thatincludes a capture domain that includes a random N-mer priming sequencefor gDNA analysis; and a barcoded oligonucleotide that includes acapture domain that can specifically bind a an analyte capture agent(e.g., an antibody with a spatial barcode), via its analyte capturesequence.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of three or more biological analytes that canbe concurrently measured include, without limitation: (a) mRNA, alineage tracing construct, and cell surface and/or intracellularproteins and/or metabolites; (b) mRNA, accessible chromatin (e.g.,ATAC-seq, DNase-seq, and/or MNase-seq), and cell surface and/orintracellular proteins and/or metabolites; (c) mRNA, genomic DNA, and aperturbation reagent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (d)mRNA, accessible chromatin, and a perturbation reagent; (e) mRNA, ananalyte capture agent (e.g., any of the MHC multimers described herein),and a perturbation reagent; (f) mRNA, cell surface and/or intracellularproteins and/or metabolites, and a perturbation agent; (g) mRNA, a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), and aperturbation reagent; (h) mRNA, an analyte capture agent, and a V(D)Jsequence of an immune cell receptor; (i) cell surface and/orintracellular proteins and/or metabolites, a an analyte capture agent(e.g., the MHC multimers described herein), and a V(D)J sequence of animmune cell receptor; (j) methylation status, mRNA, and cell surfaceand/or intracellular proteins and/or metabolites; (k) mRNA, chromatin(e.g., spatial organization of chromatin in a cell), and a perturbationreagent; (l) a V(D)J sequence of an immune cell receptor, chromatin(e.g., spatial organization of chromatin in a cell); and a perturbationreagent; and (m) mRNA, a V(D)J sequence of an immune cell receptor, andchromatin (e.g., spatial organization of chromatin in a cell), or anycombination thereof.

In some embodiments of any of the spatial analysis methods describedherein, four or more classes biological analytes can be concurrentlymeasured. A feature can be a bead that is coupled to barcoded primersthat can each participate in an assay of a different type of analyte.The feature is coupled (e.g., reversibly coupled) to a capture probethat includes a capture domain that includes a poly(T) priming sequencefor mRNA analysis and is also coupled (e.g., reversibly coupled) tocapture probe that includes a capture domain that includes a randomN-mer priming sequence for gDNA analysis. Moreover, the feature is alsocoupled (e.g., reversibly coupled) to a capture probe that binds ananalyte capture sequence of an analyte capture agent via its capturedomain. The feature can also be coupled (e.g., reversibly coupled) to acapture probe that can specifically bind a nucleic acid molecule thatcan function as a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN,zinc finger nuclease, and/or antisense oligonucleotide as describedherein), via its capture domain.

In some embodiments of any of the spatial analysis methods describedherein, each of the various spatially-barcoded capture probes present ata given feature or on a given bead include the same spatial barcodesequence. In some embodiments, each barcoded capture probe can bereleased from the feature in a manner suitable for analysis of itsrespective analyte. For example, barcoded constructs A, B, C and D canbe generated as described elsewhere herein and analyzed. Barcodedconstruct A can include a sequence corresponding to the barcode sequencefrom the bead (e.g., a spatial barcode) and a DNA sequence correspondingto a target mRNA. Barcoded construct B can include a sequencecorresponding to the barcode sequence from the bead (e.g., a spatialbarcode) and a sequence corresponding to genomic DNA. Barcoded constructC can include a sequence corresponding to the barcode sequence from thebead (e.g., a spatial barcode) and a sequence corresponding to barcodesequence associated with an analyte capture agent (e.g., an analytebinding moiety barcode). Barcoded construct D can include a sequencecorresponding to the barcode sequence from the bead (e.g., a spatialbarcode) and a sequence corresponding to a CRISPR nucleic acid (which,in some embodiments, also includes a barcode sequence). Each constructcan be analyzed (e.g., via any of a variety of sequencing methods) andthe results can be associated with the given cell from which the variousanalytes originated. Barcoded (or even non-barcoded) constructs can betailored for analyses of any given analyte associated with a nucleicacid and capable of binding with such a construct.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of four or more biological analytes that canbe concurrently measured include, without limitation: (a) mRNA, alineage tracing construct, cell surface and/or intracellular proteinsand/or metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g.,ATAC-seq, DNase-seq, and/or MNase-seq), cell surface and/orintracellular proteins and/or metabolites, and a perturbation agent(e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/orantisense oligonucleotide as described herein); (c) mRNA, cell surfaceand/or intracellular proteins and/or metabolites, an analyte captureagent (e.g., the MHC multimers described herein), and a V(D)J sequenceof an immune cell receptor (e.g., T-cell receptor); (d) mRNA, genomicDNA, a perturbation reagent, and accessible chromatin; (e) mRNA, cellsurface and/or intracellular proteins and/or metabolites, an analytecapture agent (e.g., the MHC multimers described herein), and aperturbation reagent; (f) mRNA, cell surface and/or intracellularproteins and/or metabolites, a perturbation reagent, and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor); (g) mRNA, aperturbation reagent, an analyte capture agent (e.g., the MHC multimersdescribed herein), and a V(D)J sequence of an immune cell receptor(e.g., T-cell receptor); (h) mRNA, chromatin (e.g., spatial organizationof chromatin in a cell), and a perturbation reagent; (i) a V(D)Jsequence of an immune cell receptor, chromatin (e.g., spatialorganization of chromatin in a cell); and a perturbation reagent; (j)mRNA, a V(D)J sequence of an immune cell receptor, chromatin (e.g.,spatial organization of chromatin in a cell), and genomic DNA; (k) mRNA,a V(D)J sequence of an immune cell receptor, chromatin (e.g., spatialorganization of chromatin in a cell), and a perturbation reagent, or anycombination thereof.

(b) Construction of Spatial Arrays for Multi-Analyte Analysis

This disclosure also provides methods and materials for constructing aspatial array capable of multi-analyte analysis. In some embodiments, aspatial array includes a plurality of features on a substrate where oneor more members of the plurality of features include a plurality ofoligonucleotides having a first type functional sequence andoligonucleotides having a second, different type of functional sequence.In some embodiments, a feature can include oligonucleotides with twotypes of functional sequences. A feature can be coupled tooligonucleotides comprising a TruSeq functional sequence and also tooligonucleotides comprising a Nextera functional sequence. In someembodiments, a functional sequence can include can include a sequencerspecific flow cell attachment sequence, e.g., a P5 sequence, as well asfunctional sequence, which can include sequencing primer sequences,e.g., a R1 primer binding site. In some embodiments, one or more membersof the plurality of features comprises both types of functionalsequences. In some embodiments, one or more members of the pluralityfeatures includes a first type of functional sequence. In someembodiments, one or more members of the plurality of features includes asecond type of functional sequence. In some embodiments, an additionaloligonucleotide can be added to the functional sequence to generate afull oligonucleotide where the full oligonucleotide includes a spatialbarcode sequence, an optional UMI sequence, a priming sequence, and acapture domain. Attachment of these sequences can be via ligation(including via splint ligation as is described in U.S. PatentApplication Publication No. 20140378345, the entire contents of whichare incorporated herein by reference), or any other suitable route. Asdiscussed herein, oligonucleotides can be hybridized with splintsequences that can be helpful in constructing complete fulloligonucleotides (e.g., oligonucleotides that are capable of spatialanalysis).

In some embodiments, the oligonucleotides that hybridize to thefunctional sequences (e.g., TruSeq and Nextera) located on the featuresinclude capture domains capable of capturing different types of analytes(e.g., mRNA, genomic DNA, cell surface proteins, or accessiblechromatin). In some examples, oligonucleotides that can bind to theTruSeq functional sequences can include capture domains that includepoly(T) capture sequences. In addition to the poly(T) capture sequences,the oligonucleotides that can bind the TruSeq functional groups can alsoinclude a capture domain that includes a random N-mer sequence forcapturing genomic DNA (e.g., or any other sequence or domain asdescribed herein capable of capturing any of the biological analytesdescribed herein). In such cases, the spatial arrays can be constructedby applying ratios of TruSeq-poly(T) and TruSeq-N-mer oligonucleotidesto the features comprising the functional TruSeq sequences. This canproduce spatial arrays where a portion of the oligonucleotides cancapture mRNA and a different portion of oligonucleotides can capturegenomic DNA. In some embodiments, one or more members of a plurality offeatures include both TruSeq and Nextera functional sequences. In suchcases, a feature including both types of functional sequences is capableof binding oligonucleotides specific to each functional sequence. Forexample, an oligonucleotide capable of binding to a TruSeq functionalsequence could be used to deliver an oligonucleotide including a poly(T)capture domain and an oligonucleotide capable of binding to a Nexterafunctional sequence could be used to deliver an oligonucleotideincluding an N-mer capture domain for capturing genomic DNA. It will beappreciated by a person of ordinary skill in the art that anycombination of capture domains (e.g., capture domains having any of thevariety of capture sequences described herein capable of binding to anyof the different types of analytes as described herein) could becombined with oligonucleotides capable of binding to TruSeq and Nexterafunctional sequences to construct a spatial array.

In some embodiments, an oligonucleotide that includes a capture domain(e.g., an oligonucleotide capable of coupling to an analyte) or ananalyte capture agent can include an oligonucleotide sequence that iscapable of binding or ligating to an assay primer. The adapter can allowthe capture probe or the analyte capture agent to be attached to anysuitable assay primers and used in any suitable assays. The assay primercan include a priming region and a sequence that is capable of bindingor ligating to the adapter. In some embodiments, the adapter can be anon-specific primer (e.g., a 5′ overhang) and the assay primer caninclude a 3′ overhang that can be ligated to the 5′ overhang. Thepriming region on the assay primer can be any primer described herein,e.g., a poly (T) primer, a random N-mer primer, a target-specificprimer, or an analyte capture agent capture sequence.

In some examples, an oligonucleotide can includes an adapter, e.g., a 5′overhang with 10 nucleotides. The adapter can be ligated to assayprimers, each of which includes a 3′ overhang with 10 nucleotides thatcomplementary to the 5′ overhang of the adapter. The capture probe canbe used in any assay by attaching to the assay primer designed for thatassay.

Adapters and assay primers can be used to allow the capture probe or theanalyte capture agent to be attached to any suitable assay primers andused in any suitable assays. A capture probe that includes a spatialbarcode can be attached to a bead that includes a poly(dT) sequence. Acapture probe including a spatial barcode and a poly(T) sequence can beused to assay multiple biological analytes as generally described herein(e.g., the biological analyte includes a poly(A) sequence or is coupledto or otherwise is associated with an analyte capture agent comprising apoly(A) sequence as the analyte capture sequence).

A splint oligonucleotide with a poly(A) sequence can be used tofacilitate coupling to a capture probe that includes a spatial barcodeand a second sequence that facilitates coupling with an assay primer.Assay primers include a sequence complementary to the splint oligosecond sequence and an assay-specific sequence that determines assayprimer functionality (e.g., a poly(T) primer, a random N-mer primer, atarget-specific primer, or an analyte capture agent capture sequence asdescribed herein).

In some embodiments of any of the spatial profiling methods describedherein, a feature can include a capture probe that includes a spatialbarcode comprising a switch oligonucleotide, e.g., with a 3′ end 3rG.For example, a feature (e.g., a gel bead) with a spatial barcodefunctionalized with a 3rG sequence can be used that enables templateswitching (e.g., reverse transcriptase template switching), but is notspecific for any particular assay. In some embodiments, the assayprimers added to the reaction can determine which type of analytes areanalyzed. For example, the assay primers can include binding domainscapable of binding to target biological analytes (e.g., poly (T) formRNA, N-mer for genomic DNA, etc.). A capture probe (e.g., anoligonucleotide capable of spatial profiling) can be generated by usinga reverse transcriptase enzyme/polymerase to extend, which is followedby template switching onto the barcoded adapter oligonucleotide toincorporate the barcode and other functional sequences. In someembodiments, the assay primers include capture domains capable ofbinding to a poly(T) sequence for mRNA analysis, random primers forgenomic DNA analysis, or a capture sequence that can bind a nucleic acidmolecule coupled to an analyte binding moiety (e.g., a an analytecapture sequence of an analyte capture agent) or a nucleic acid moleculethat can function in as a perturbation reagent (e.g., a CRISPRcrRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein).

V. Systems for Sample Analysis

The methods described above for analyzing biological samples can beimplemented using a variety of hardware components. In this section,examples of such components are described. However, it should beunderstood that in general, the various steps and techniques discussedherein can be performed using a variety of different devices and systemcomponents, not all of which are expressly set forth.

FIG. 22A is a schematic diagram showing an example sample handlingapparatus 2200. Sample handling apparatus 2200 includes a sample chamber2202 that, when closed or sealed, is fluid-tight. Within chamber 2202, afirst holder 2204 holds a first substrate 2206 on which a sample 2208 ispositioned. Sample chamber 2202 also includes a second holder 2210 thatholds a second substrate 2212 with an array of features 2214, asdescribed above.

A fluid reservoir 2216 is connected to the interior volume of samplechamber 2202 via a fluid inlet 2218. Fluid outlet 2220 is also connectedto the interior volume of sample chamber 2202, and to valve 2222. Inturn, valve 2222 is connected to waste reservoir 2224 and, optionally,to analysis apparatus 2226. A control unit 2228 is electricallyconnected to second holder 2210, to valve 2222, to waste reservoir 2224,and to fluid reservoir 2216.

During operation of apparatus 2200, any of the reagents, solutions, andother biochemical components described above can be delivered intosample chamber 2202 from fluid reservoir 2216 via fluid inlet 2218.Control unit 2228, connected to fluid reservoir 2216, can control thedelivery of reagents, solutions, and components, and adjust the volumesand flow rates according to programmed analytical protocols for varioussample types and analysis procedures. In some embodiments, fluidreservoir 2216 includes a pump, which can be controlled by control unit2228, to facilitate delivery of substances into sample chamber 2202.

In certain embodiments, fluid reservoir 2216 includes a plurality ofchambers, each of which is connected to fluid inlet 2218 via a manifold(not shown). Control unit 2228 can selectively deliver substances fromany one or more of the multiple chambers into sample chamber 2202 byadjusting the manifold to ensure that the selected chambers arefluidically connected to fluid inlet 2218.

In general, control unit 2228 can be configured to introduce substancesfrom fluid reservoir 2216 into sample chamber 2202 before, after, orboth before and after, sample 2208 on first substrate 2206 hasinteracted with the array of features 2214 on first substrate 2212. Manyexamples of such substances have been described previously. Examples ofsuch substances include, but are not limited to, permeabilizing agents,buffers, fixatives, staining solutions, washing solutions, and solutionsof various biological reagents (e.g., enzymes, peptides,oligonucleotides, primers).

To initiate interaction between sample 2208 and feature array 2214, thesample and array are brought into spatial proximity. To facilitate thisstep, second holder 2210—under the control of control unit 2228—cantranslate second substrate 2212 in any of the x-, y-, and z-coordinatedirections. In particular, control unit 2228 can direct second holder2210 to translate second substrate 2212 in the z-direction so thatsample 2208 contacts, or nearly contacts, feature array 2214.

In some embodiments, apparatus 2200 can optionally include an alignmentsub-system 2230, which can be electrically connected to control unit2228. Alignment sub-system 2230 functions to ensure that sample 2208 andfeature array 2214 are aligned in the x-y plane prior to translatingsecond substrate 2212 in the z-direction so that sample 2208 contacts,or nearly contacts, feature array 2214.

Alignment sub-system 2230 can be implemented in a variety of ways. Insome embodiments, for example, alignment sub-system 2230 includes animaging unit that obtains one or more images showing fiducial markingson first substrate 2206 and/or second substrate 2212. Control unit 2218analyzes the image(s) to determine appropriate translations of secondsubstrate 2212 in the x- and/or y-coordinate directions to ensure thatsample 2208 and feature array 2214 are aligned prior to translation inthe z-coordinate direction.

In certain embodiments, control unit 2228 can optionally regulate theremoval of substances from sample chamber 2202. For example, controlunit 2228 can selectively adjust valve 2222 so that substancesintroduced into sample chamber 2202 from fluid reservoir 2216 aredirected into waste reservoir 2224. In some embodiments, waste reservoir2224 can include a reduced-pressure source (not shown) electricallyconnected to control unit 2228. Control unit 2228 can adjust the fluidpressure in fluid outlet 2220 to control the rate at which fluids areremoved from sample chamber 2202 into waste reservoir 2224.

In some embodiments, analytes from sample 2208 or from feature array2214 can be selectively delivered to analysis apparatus 2226 viasuitable adjustment of valve 2222 by control unit 2228. As describedabove, in some embodiments, analysis apparatus 2226 includes areduced-pressure source (not shown) electrically connected to controlunit 2228, so that control unit 2228 can adjust the rate at whichanalytes are delivered to analysis apparatus 2226. As such, fluid outlet2220 effectively functions as an analyte collector, while analysis ofthe analytes is performed by analysis apparatus 2226. It should be notedthat not all of the workflows and methods described herein areimplemented via analysis apparatus 2226. For example, in someembodiments, analytes that are captured by feature array 2214 remainbound to the array (i.e., are not cleaved from the array), and featurearray 2214 is directly analyzed to identify specifically-bound samplecomponents.

In addition to the components described above, apparatus 2200 canoptionally include other features as well. In some embodiments, forexample, sample chamber 2202 includes a heating sub-system 2232electrically connected to control unit 2228. Control unit 2228 canactivate heating sub-system 2232 to heat sample 2208 and/or featurearray 2214, which can help to facilitate certain steps of the methodsdescribed herein.

In certain embodiments, sample chamber 2202 includes an electrode 2234electrically connected to control unit 2228. Control unit 2228 canoptionally activate electrode 2234, thereby establishing an electricfield between the first and second substrates. Such fields can be used,for example, to facilitate migration of analytes from sample 2208 towardfeature array 2214.

In some of the methods described herein, one or more images of a sampleand/or a feature array are acquired. Imaging apparatus that is used toobtain such images can generally be implemented in a variety of ways.FIG. 22B shows one example of an imaging apparatus 2250. Imagingapparatus 2250 includes a light source 2252, light conditioning optics2254, light delivery optics 2256, light collection optics 2260, lightadjusting optics 2262, and a detection sub-system 2264. Each of theforegoing components can optionally be connected to control unit 2228,or alternatively, to another control unit. For purposes of explanationbelow, it will be assumed that control unit 2228 is connected to thecomponents of imaging apparatus 2250.

During operation of imaging apparatus 2250, light source 2252 generateslight. In general, the light generated by source 2252 can include lightin any one or more of the ultraviolet, visible, and/or infrared regionsof the electromagnetic spectrum. A variety of different light sourceelements can be used to generate the light, including (but not limitedto) light emitting diodes, laser diodes, laser sources, fluorescentsources, incandescent sources, and glow-discharge sources.

The light generated by light source 2252 is received by lightconditioning optics 2254. In general, light conditioning optics 2254modify the light generated by light source 2252 for specific imagingapplications. For example, in some embodiments, light conditioningoptics 2254 modify the spectral properties of the light, e.g., byfiltering out certain wavelengths of the light. For this purpose, lightconditioning optics 2254 can include a variety of spectral opticalelements, such as optical filters, gratings, prisms, and chromatic beamsplitters.

In certain embodiments, light conditioning optics 2254 modify thespatial properties of the light generated by light source 2252. Examplesof components that can be used for this purpose include (but are notlimited to) apertures, phase masks, apodizing elements, and diffusers.

After modification by light conditioning optics 2254, the light isreceived by light delivery optics 2256 and directed onto sample 2208 orfeature array 2214, either of which is positioned on a mount 2258. Lightconditioning optics 2254 generally function to collect and direct lightonto the surface of the sample or array. A variety of different opticalelements can be used for this purpose, and examples of such elementsinclude, but are not limited to, lenses, mirrors, beam splitters, andvarious other elements having non-zero optical power.

Light emerging from sample 2208 or feature array 2214 is collected bylight collection optics 2260. In general, light collection optics 2260can include elements similar to any of those described above inconnection with light delivery optics 2256. The collected light can thenoptionally be modified by light adjusting optics 2262, which cangenerally include any of the elements described above in connection withlight conditioning optics 2254.

The light is then detected by detection sub-system 2264. Generally,detection sub-system 2264 functions to generate one or more images ofsample 2208 or feature array 2214 by detecting light from the sample orfeature array. A variety of different imaging elements can be used indetection sub-system 2264, including CCD detectors and other imagecapture devices.

Each of the foregoing components can optionally be connected to controlunit 2228 as shown in FIG. 22B, so that control unit 2228 can adjustvarious properties of the imaging apparatus. For example, control unit2228 can adjust the position of sample 2208 or feature array 2214relative to the position of the incident light, and also with respect tothe focal plane of the incident light (if the incident light isfocused). Control unit 2228 can also selectively filter both theincident light and the light emerging from the sample.

Imaging apparatus 2250 can typically obtain images in a variety ofdifferent imaging modalities. In some embodiments, for example, theimages are transmitted light images, as shown in FIG. 22B. In certainembodiments, apparatus 2250 is configured to obtain reflection images.In some embodiments, apparatus 2250 can be configured to obtainbirefringence images, fluorescence images, phosphorescence images,multiphoton absorption images, and more generally, any known image type.

In general, control unit 2228 can perform any of the method stepsdescribed herein that do not expressly require user intervention bytransmitting suitable control signals to the components of samplehandling apparatus 2200 and/or imaging apparatus 2250. To perform suchsteps, control unit 2228 generally includes software instructions that,when executed, cause control unit 2228 to undertake specific steps. Insome embodiments, control unit 2228 includes an electronic processor andsoftware instructions that are readable by the electronic processor, andcause the processor to carry out the steps describe herein. In certainembodiments, control unit 2228 includes one or more application-specificintegrated circuits having circuit configurations that effectivelyfunction as software instructions.

Control unit 2228 can be implemented in a variety of ways. FIG. 22C is aschematic diagram showing one example of control unit 2228, including anelectronic processor 2280, a memory unit 2282, a storage device 2284,and an input/output interface 2286. Processor 2280 is capable ofprocessing instructions stored in memory unit 2282 or in storage device2284, and to display information on input/output interface 2286.

Memory unit 2282 stores information. In some embodiments, memory unit2282 is a computer-readable medium. Memory unit 2282 can includevolatile memory and/or non-volatile memory. Storage device 2284 iscapable of providing mass storage, and in some embodiments, is acomputer-readable medium. In certain embodiments, storage device 2284may be a floppy disk device, a hard disk device, an optical disk device,a tape device, a solid state device, or another type of writeablemedium.

The input/output interface 2286 implements input/output operations. Insome embodiments, the input/output interface 2286 includes a keyboardand/or pointing device. In some embodiments, the input/output interface2286 includes a display unit for displaying graphical user interfacesand/or display information.

Instructions that are executed and cause control unit 2228 to performany of the steps or procedures described herein can be implemented indigital electronic circuitry, or in computer hardware, firmware, or incombinations of these. The instructions can be implemented in a computerprogram product tangibly embodied in an information carrier, e.g., in amachine-readable storage device, for execution by a programmableprocessor (e.g., processor 2280). The computer program can be written inany form of programming language, including compiled or interpretedlanguages, and it can be deployed in any form, including as astand-alone program or as a module, component, subroutine, or other unitsuitable for use in a computing environment. Storage devices suitablefor tangibly embodying computer program instructions and data includeall forms of non-volatile memory, including by way of examplesemiconductor memory devices, such as EPROM, EEPROM, and flash memorydevices; magnetic disks such as internal hard disks and removable disks;magneto-optical disks; and CD-ROM and DVD-ROM disks. The processor andthe memory can be supplemented by, or incorporated in, ASICs(application-specific integrated circuits).

Processor 2280 can include any one or more of a variety of suitableprocessors. Suitable processors for the execution of a program ofinstructions include, by way of example, both general and specialpurpose microprocessors, and the sole processor or one of multipleprocessors of any kind of computer or computing device.

Exemplary Embodiments

In some non-limiting examples of the workflows described herein, thesample can be immersed in 100% chilled methanol and incubated for 30minutes at −20° C. After 20 minutes, the sample can be removed andrinsed in ultrapure water. After rinsing the sample, fresh eosinsolution is prepared, and the sample can be covered in isopropanol.After incubating the sample in isopropanol for 1 minute, the reagent canbe removed by holding the slide at an angle, where the bottom edge ofthe slide can be in contact with a laboratory wipe and air dried. Thesample can be uniformly covered in hematoxylin solution and incubatedfor 7 minutes at room temperature. After incubating the sample inhematoxylin for 7 minutes, the reagent can be removed by holding theslide at an angle, where the bottom edge of the slide can be in contactwith a laboratory wipe. The slide containing the sample can be immersedin water and the excess liquid can be removed. After that, the samplecan be covered with bluing buffer and can be incubated for 2 minutes atroom temperature. The slide containing the sample can again be immersedin water, and uniformly covered with eosin solution and incubated for 1minute at room temperature. The slide can be air-dried for no more than30 minutes and incubated for 5 minutes at 37° C. The sample can beimaged using brightfield imaging setting.

Further, the biological sample can be processed by the followingexemplary steps for sample permeabilization and cDNA generation. Thesample can be exposed to a permeabilization enzyme and incubated at 37°C. for the pre-determined permeabilization time (which is tissue typespecific). The permeabilization enzyme can be removed and the sampleprepared for analyte capture by adding 0.1×SSC buffer. The sample canthen subjected to a pre-equilibration thermocycling protocol (e.g., lidtemperature and pre-equilibrate at 53° C., reverse transcription at 53°C. for 45 minutes, and then hold at 4° C.) and the SSC buffer can beremoved. A Master Mix, containing nuclease-free water, a reversetranscriptase reagent, a template switch oligo, a reducing agent, and areverse transcriptase enzyme can be added to the biological sample andsubstrate, and the sample with the Master Mix can be subjected to athermocycling protocol (e.g., perform reverse transcription at 53° C.for 45 minutes and hold at 4° C.). Second strand synthesis can beperformed on the substrate by subjecting the substrate to athermocycling protocol (e.g., pre-equilibrate at 65° C., second strandsynthesis at 65° C. for 15 minutes, then hold at 4° C.). The Master Mixreagents can be removed from the sample and 0.8M KOH can be applied andincubated for 5 minutes at room temperature. The KOH can be removed andelution buffer can be added and removed from the sample. A Second StrandMix, including a second strand reagent, a second strand primer, and asecond strand enzyme, can be added to the sample and the sample can besealed and incubated. At the end of the incubation, the reagents can beremoved and elution buffer can be added and removed from the sample, and0.8M KOH can be added again to the sample and the sample can beincubated for 10 minutes at room temperature. Tris-HCl can be added andthe reagents can be mixed. The sample can be transferred to a new tube,vortexed, and placed on ice.

Further the biological sample can be processed by the followingexemplary steps for cDNA amplification and quality control. A qPCR Mix,including nuclease-free water, qPCR Master Mix, and cDNA primers, can beprepared and pipetted into wells in a qPCR plate. A small amount ofsample can be added to the plated qPCR Mix, and thermocycled accordingto a predetermined thermocycling protocol (e.g., step 1: 98° C. for 3minutes, step 2: 98° C. for 5 seconds, step 3: 63° C. for 30 seconds,step 4: record amplification signal, step 5: repeating 98° C. for 5seconds, 63° C. for 30 seconds for a total of 25 cycles). Aftercompleting the thermocycling, a cDNA amplification mix, includingamplification mix and cDNA primers, can be prepared and combined withthe remaining sample and mixed. The sample can then be incubated andthermocycled (e.g., lid temperature at 105° C. for ˜45-60 minutes; step1: 98° C. for 3 minutes, step 2: 98° C. for 15 seconds, step 3: 63° C.for 20 seconds, step 4: 72° C. for one minute, step 5: [the number ofcycles determined by qPCR Cq Values], step 6: 72° C. for 1 minute, andstep 7: hold at 4° C.). The sample can then be stored at 4° C. for up to72 hours or at −20° C. for up to 1 week, or resuspended in0.6×SPRIselect Reagent and pipetted to ensure proper mixing. The samplecan then be incubated at 5 minutes at room temperature, and cleared byplacing the sample on a magnet (e.g., the magnet is in the highposition). The supernatant can be removed and 80% ethanol can be addedto the pellet, and incubated for 30 seconds. The ethanol can be removedand the pellet can be washed again. The sample can then be centrifugedand placed on a magnet (e.g., the magnet is on the low position). Anyremaining ethanol can be removed and the sample can be air dried for upto 2 minutes. The magnet can be removed and elution buffer can be addedto the sample, mixed, and incubated for 2 minutes at room temperature.The sample can then be placed on the magnet (e.g., on low position)until the solution clears. The sample can be transferred to a new tubestrip and stored at 4° C. for up to 72 hours or at −20° C. for up to 4weeks. A portion of the sample can be run on an Agilent Bioanalyzer HighSensitivity chip, where a region can be selected and the cDNAconcentration can be measured to calculate the total cDNA yield.Alternatively, the quantification can be determined by AgilentBioanalyzer or Agilent TapeStation.

Further, the biological sample can be processed by the followingexemplary steps for spatial gene expression library construction. AFragmentation Mix, including a fragmentation buffer and fragmentationenzyme, can be prepared on ice. Elution buffer and fragmentation mix canbe added to each sample, mixed, and centrifuged. The sample mix can thenbe placed in a thermocycler and cycled according to a predeterminedprotocol (e.g., lid temperature at 65° C. for ˜35 minutes, pre-coolblock down to 4° C. before fragmentation at 32° C. for 5 minutes,End-repair and A-tailing at 65° C. for 30 minutes, and holding at 4°C.). The 0.6× SPRIselect Reagent can be added to the sample andincubated at 5 minutes at room temperature. The sample can be placed ona magnet (e.g., in the high position) until the solution clears, and thesupernatant can be transferred to a new tube strip. 0.8×SPRIselectReagent can be added to the sample, mixed, and incubated for 5 minutesat room temperature. The sample can be placed on a magnet (e.g., in thehigh position) until the solution clears. The supernatant can be removedand 80% ethanol can be added to the pellet, the pellet can be incubatedfor 30 seconds, and the ethanol can be removed. The ethanol wash can berepeated and the sample placed on a magnet (e.g., in the low position)until the solution clears. The remaining ethanol can be removed andelution buffer can be added to the sample, mixed, and incubated for 2minutes at room temperature. The sample can be placed on a magnet (e.g.,in the high position) until the solution clears, and a portion of thesample can be moved to a new tube strip. An Adaptor Ligation Mix,including ligation buffer, DNA ligase, and adaptor oligos, can beprepared and centrifuged. The Adaptor Ligation Mix can be added to thesample, pipette-mixed, and centrifuged briefly. The sample can then bethermocycled according to a predetermined protocol (e.g., lidtemperature at 30° C. for ˜15 minutes, step 1: 20° C. for 15 minutes,step 2: 4° C. hold). The sample can be vortexed to re-suspend SPRIselectReagent, additional 0.8×SPRIselect Reagent can be added to the sampleand incubated for 5 minutes at room temperature, and placed on a magnet(e.g., in the high position) until the solution clears. The supernatantcan be removed and the pellet can be washed with 80% ethanol, incubatedfor 30 seconds, and the ethanol can be removed. The ethanol wash can berepeated, and the sample can be centrifuged briefly before placing thesample on a magnet (e.g., in the low position). Any remaining ethanolcan be removed and the sample can be air dried for a maximum of 2minutes. The magnet can be removed, and elution buffer can be added tothe sample, and the sample can be pipette-mixed, incubated for 2 minutesat room temperature, and placed on a magnet (e.g., in the low position)until the solution clears. A portion of the sample can be transferred toa new tube strip. Amplification mix, can be prepared and combined withthe sample. An individual Dual Index TT Set A can be added to thesample, pipette-mixed and subjected to a pre-determined thermocyclingprotocol (e.g., lid temperature at 105° C. for ˜25-40 minutes, step 1:98° C. for 45 seconds, step 2: 98° C. for 20 seconds, step 3: 54° C. for30 seconds; step 4: 72° C. for 20 seconds, step 5: reverting to step 2for a predetermined number of cycles, step 6: 72° C. for 1 minute, and4° C. on hold). Vortex to re-suspend the SPRIselect Reagent, additional0.6×SPRIselect Reagent can be added to each sample, mixed, and incubatedfor 5 minutes at room temperature. The sample can be placed on a magnet(e.g., in the high position) until the solution clears, and thesupernatant can be transferred to a new tube strip. The 0.8×SPRIselectReagent can be added to each sample, pipette-mixed, and incubated for 5minutes at room temperature. The sample can then be placed on a magnet(e.g., in the high position) until the solution clears. The supernatantcan be removed, and the pellet can be washed with 80% ethanol, incubatedfor 30 seconds, and then the ethanol can be removed. The ethanol washcan be repeated, the sample centrifuged, and placed on a magnet (e.g.,in the low position) to remove any remaining ethanol. The sample can beremoved from the magnet and Elution Buffer can be added to the sample,pipette-mixed, and incubated at 2 minutes at room temperature. Thesample can be placed on a magnet (e.g., in the low position) until thesolution clears and a portion of the sample can be transferred to a newtube strip. The sample can be stored at 4° C. for up to 72 hours, or at−20° C. for long-term storage. The average fragment size can bedetermined using a Bioanalyzer trace or an Agilent TapeStation.

The library can be sequenced using available sequencing platforms,including, MiSeq, NextSeq 500/550, HiSeq 2500, HiSeq 3000/4000, NovaSeq,and iSeq.

In alternate embodiments of the above described workflows, a biologicalsample can be permeabilized by exposing the sample to greater than about1.0 w/v % (e.g., greater than about 2.0 w/v %, greater than about 3.0w/v %, greater than about 4.0 w/v %, greater than about 5.0 w/v %,greater than about 6.0 w/v %, greater than about 7.0 w/v %, greater thanabout 8.0 w/v %, greater than about 9.0 w/v %, greater than about 10.0w/v %, greater than about 11.0 w/v %, greater than about 12.0 w/v %, orgreater than about 13.0 w/v %) sodium dodecyl sulfate (SDS). In someembodiments, a biological sample can be permeabilized by exposing thesample (e.g., for about 5 minutes to about 1 hour, about 5 minutes toabout 40 minutes, about 5 minutes to about 30 minutes, about 5 minutesto about 20 minutes, or about 5 minutes to about 10 minutes) to about1.0 w/v % to about 14.0 w/v % (e.g., about 2.0 w/v % to about 14.0 w/v%, about 2.0 w/v % to about 12.0 w/v %, about 2.0 w/v % to about 10.0w/v %, about 4.0 w/v % to about 14.0 w/v %, about 4.0 w/v % to about12.0 w/v %, about 4.0 w/v % to about 10.0 w/v %, about 6.0 w/v % toabout 14.0 w/v %, about 6.0 w/v % to about 12.0 w/v %, about 6.0 w/v %to about 10.0 w/v %, about 8.0 w/v % to about 14.0 w/v %, about 8.0 w/v% to about 12.0 w/v %, about 8.0 w/v % to about 10.0 w/v %, about 10.0%w/v % to about 14.0 w/v %, about 10.0 w/v % to about 12.0 w/v %, orabout 12.0 w/v % to about 14.0 w/v %) SDS and/or proteinase K (e.g., ata temperature of about 35° C. to about 50° C., about 35° C. to about 45°C., about 35° C. to about 40° C., about 40° C. to about 50° C., about40° C. to about 45° C., or about 45° C. to about 50° C.).

In alternate embodiments of the above described workflows, the substratecan include 5000 features (e.g., spots, beads, pads, etc.). For example,a substrate including 5,000 features can provide higher resolution thanan array including 1,000 features (e.g., spots, beads, pads, etc.). Inalternate embodiments of the above described workflows, the substratecan include fiducial markers. For example, fiducial markers can allowfor proper orientation, detection, and/or rotation of the sample on thesubstrate.

In alternative embodiments of the above described workflows, one or morespecies of RNA (e.g., ribosomal, mitochondrial RNA, etc.) can bedown-selected (e.g., removed or depleted). Non-limiting examples ofhybridization and capture method ribosomal RNA depletion includeRiboMinus™, RiboCop™, and Ribo-Zero™. Another non-limiting RNA depletionmethod involves hybridization of complementary DNA oligonucleotides tounwanted RNA followed by degradation of the RNA/DNA hybrids using RNaseH. Non-limiting examples of a hybridization and degradation methodinclude NEBNext® rRNA depletion, NuGEN AnyDeplete, and RiboZero Plus.Another non-limiting ribosomal RNA depletion method includes ZapR™digestion, for example SMARTer. In the SMARTer method, random nucleicacid adapters are hybridized to RNA for first-strand synthesis andtailing by reverse transcriptase, followed by template switching andextension by reverse transcriptase.

In alternative embodiments of the above described workflows, additionalreagents can be added to improve the recovery of one or more targetmolecules (e.g., cDNA molecules, mRNA transcripts). For example,addition of carrier RNA to a RNA sample workflow process can increasethe yield of extracted RNA/DNA hybrids from the biological sample.Carrier molecules (e.g., RNA) can be useful when the concentration ofinput or target molecules is low as compared to remaining molecules.Generally, single target molecules cannot form a precipitate, andaddition of the carrier molecules can help in forming a precipitate.Carrier RNA can be added immediately prior to a second strand synthesisstep. Carrier RNA can be added immediately prior to a second strand cDNAsynthesis on oligonucleotides (e.g., via denaturation) released from thesubstrate. Carrier RNA can be added immediately prior to a post in vitrotranscription clean-up step. Carrier RNA can be added prior to amplifiedRNA purification and quantification. Carrier RNA can be added before RNAquantification. Carrier RNA can be added immediately prior to both asecond strand cDNA synthesis step and a post in vitro transcriptionclean-up step.

In alternative embodiments of the above described workflows,permeabilization can be performed with acetone (e.g., with fresh frozentissue). When acetone fixation is performed, pre-permeabilization stepsmay not be performed. Alternatively, acetone fixation can be performedwith permeabilization steps. In alternative embodiments of the abovedescribed workflow, PAS staining can be performed when samples arepermeabilized with acetone.

In non-limiting examples of any of the workflows described herein, anucleic acid molecule is produced that includes a contiguous nucleotidesequence comprising: (a) a first primer sequence (e.g., Read 1); (b) aspatial barcode; (c) a unique molecular sequence (UMI); (d) a capturedomain; (e) a sequence complementary to a sequence present in a nucleicacid from a biological sample; (f) a second primer sequence (e.g., Read2) that is substantially complementary to a sequence of a templateswitching oligonucleotide (TSO). In some embodiments of these nucleicacid molecules, the nucleic acid molecule is a single-stranded nucleicacid molecule. In some embodiments of these nucleic acid molecules, thenucleic acid molecule is a double-stranded nucleic acid molecule. Insome embodiments of these nucleic acid molecules, (a) through (f) arepositioned in a 5′ to 3′ direction in the contiguous nucleotidesequence. In some embodiments of any of these nucleic acid molecules,the nucleic acid molecule is attached to a substrate (e.g., a slide). Insome embodiments of any of these nucleic acid molecules, the 5′ end ofthe contiguous nucleic acid sequence is attached to the substrate (e.g.,a slide). In some embodiments of any of these nucleic acid molecules,the contiguous nucleotide sequence is a chimeric RNA and DNA sequence.In some embodiments of any of these nucleic acid molecules, thecontiguous nucleotide sequence is a DNA sequence.

In non-limiting examples of any of the workflows described herein, anucleic acid molecule is produced that includes a contiguous nucleotidesequence comprising: (a) a sequence complementary to a first primersequence (e.g., a sequence complementary to Read 1); (b) a sequencecomplementary to a spatial barcode; (c) a sequence complementary to aunique molecular sequence; (d) a sequence complementary to a capturedomain; (e) a sequence present in a nucleic acid from a biologicalsample; and (f) a sequence of a template switching oligonucleotide(TSO). In some embodiments of any of these nucleic acid molecules, thenucleic acid molecule is single-stranded. In some embodiments of any ofthese nucleic acid molecules, the nucleic acid molecule isdouble-stranded. In some embodiments of any of these nucleic acidmolecules, the contiguous nucleotide sequence is a DNA sequence. In someembodiments of any of these nucleic acid molecules, (a) through (f) arepositioned in a 3′ to 5′ direction in the contiguous nucleotidesequence.

In non-limiting examples of any of the workflows described herein, anucleic acid molecule is produced that includes a contiguous nucleotidesequence comprising: (a) a first primer sequence (e.g., Read 1); (b) aspatial barcode; (c) a unique molecular sequence (UMI); (d) a capturedomain; (e) a sequence complementary to a sequence present in a nucleicacid from a biological sample; and (f) a second primer sequence (Read2). In some embodiments of any of these nucleic acid molecules, thenucleic acid molecule is a single-stranded nucleic acid molecule. Insome embodiments of any of these nucleic acid molecules, the nucleicacid molecule is a double-stranded nucleic acid molecule. In someembodiments of any of these nucleic acid molecules, (a) through (f) arepositioned in a 5′ to 3′ direction in the contiguous nucleotidesequence. In some embodiments of any of these nucleic acid molecules,the contiguous nucleotide sequence is a DNA sequence. In someembodiments of any of these nucleic acid molecules, the contiguousnucleotide sequence further comprises 3′ to (f): (g) a sequencecomplementary to a first adaptor sequence; and (h) a sequencecomplementary to a third primer sequence. In some embodiments of any ofthe nucleic acid molecules, the first adaptor sequence is an i7 sampleindex sequence. In some embodiments of any of these nucleic acidmolecules, the third primer sequence is a P7 primer sequence. In someembodiments of any of these nucleic acid molecules, (h) is 3′ positionedrelative to (g) in the contiguous nucleotide sequence. In someembodiments of any of these nucleic acid molecules, the contiguousnucleotide sequence further comprises 5′ to (a): (i) a second adaptorsequence; and (ii) a fourth primer sequence. In some embodiments of anyof these nucleic acid molecules, the second adaptor sequence is an i5sample index sequence. In some embodiments of any of these nucleic acidmolecules, the fourth primer sequence is a P5 primer sequence. In someembodiments of any of these nucleic acid molecules, (ii) is 5′positioned relative to (i) in the contiguous nucleotide sequence.

In non-limiting examples of any of the workflows described herein, anucleic acid molecule is produced that includes a contiguous nucleotidesequence comprising: (a) a sequence complementary to a first primersequence; (b) a sequence complementary to a spatial barcode; (c) asequence complementary to a unique molecular sequence; (d) a sequencecomplementary to a capture domain; (e) a sequence present in a nucleicacid from a biological sample; and (f) a sequence complementary to asecond primer sequence. In some embodiments of these nucleic acidmolecules, a sequence complementary to a first primer sequence is asequence complementary to Read 1. In some embodiments of these nucleicacid molecules, a sequence complementary to a second primer sequence isa sequence complementary to Read 2. In some embodiments of any of thesenucleic acid molecules, the nucleic acid molecule is a single-strandednucleic acid molecule. In some embodiments of any of these nucleic acidmolecules, the nucleic acid molecule is a double-stranded nucleic acidmolecule. In some embodiments of any of these nucleic acid molecules,(a) through (f) are positioned in a 3′ to 5′ direction in the contiguousnucleotide sequence. In some embodiments of any of these nucleic acidmolecules, the contiguous nucleotide sequence is a DNA sequence. In someembodiments of any of these nucleic acid molecules, the contiguousnucleotide sequence further comprises 5′ to (f): (g) a first adaptorsequence; and (h) a third primer sequence. In some embodiments of any ofthese nucleic acid molecules, the first adaptor sequence is an i7 sampleindex sequence. In some embodiments of any of these nucleic acidmolecules, the third primer sequence is a P7 primer sequence. In someembodiments of any of these nucleic acid molecules, (h) is 5′ positionedrelative to (g) in the contiguous nucleotide sequence. In someembodiments of any of these nucleic acid molecules, the contiguousnucleotide sequence further comprises 3′ to (a): (i) a sequencecomplementary to a second adaptor sequence; and (ii) a sequencecomplementary to a fourth primer sequence. In some embodiments of any ofthese nucleic acid molecules, the second adaptor sequence is an i5sample index sequence. In some embodiments of any of these nucleic acidmolecules, the fourth primer sequence is a P5 primer sequence. In someembodiments of any of these nucleic acid molecules, (ii) is 3′positioned relative to (i) in the contiguous nucleotide sequence.

RNA Integrity Number

Provided herein is a non-limiting example of a protocol for determiningthe RNA integrity Number (RIN) in a tissue that can include collectingbreast cancer tissue and snap freezing in liquid nitrogen. Tissue can beembedded in OCT and sectioned at 10 or 12 μM thickness at −20° C. andmounted directly on a spatial array including capture probes having an18S rRNA capture domain. Tissue can be fixed and stained using aHematoxylin and Eosin (H&E) staining protocol. Briefly, Mayer'sHematoxylin can be added, washed in water, incubated in Bluing buffer,washed in water, stained with Eosin, then washed in water, and finallydried. For visualizing H&E staining, sections can be mounted with 85%glycerol and covered with a coverslip. Bright field imaging can beperformed using the Metafer Slide Scanning Platform (Metasystems) whereraw images are stitched together with the VSlide software (Metasystems).Glycerol can be removed by holding the spatial array or glass slide inwater until the coverslip falls off and then was air dry until theremaining liquid evaporates. Hematoxylin, from the H&E stain, can beoptionally removed from the tissue section, for example, by washing indilute HCl (0.01M) prior to further processing. The tissue sections arethen ready for further processing.

Following staining, the 18S rRNA present in the tissue sections arecaptured by the 18S rRNA specific capture domains on the spatial array.The 18S rRNA is then converted to cDNA in situ. Specifically, reversetranscription is performed on the spatial array in a sealedhybridization cassette by adding 70 μl reaction mixture including 1×First-strand buffer, 5 mM DTT, 1 M Betaine, 6 mM MgCl2, 1 mM dNTPs, 0.2mg/ml BSA, 50 ng/μl Actinomycin D, 10% DMSO, 20 U/μl SuperScript IIIReverse Transcriptase, 2 U/μl RNaseOUT Recombinant RibonucleaseInhibitor. The reaction is performed overnight at 42° C. overnight.After incubation cDNA synthesis mixture is removed and the tissue waswashed with 0.1×SSC buffer.

In order to prepare the spatial array for oligonucleotide probe labelingand imaging, the breast cancer tissue and rRNA is removed. Tissueremoval can be performed first by incubation with β-mercaptoethanol inRLT lysis buffer at a 3:100 ratio at 56° C. for 1 hour with continuousshaking at 300 rpm. All tissues can be incubated with a 1:7 ratio ofProteinase K and PKD buffer for 1 hour at 56° C. using short intervalswith gentle shaking at 300 rpm. The spatial array is then washed withcontinuous shaking at 300 rpm as follows: first in 2×SSC with 0.1% SDSat 50° C. for 10 min, then in 0.2×SSC at RT for 1 min and finally in0.1×SSC at RT for 1 min. The spatial array is then spin-dried and putback into the hybridization cassette. rRNA removal can be performedusing a reaction mixture containing the following final concentrations:1× First-strand buffer, 0.4 mg/ml BSA and 16.3 mU/μl RNase H. Thereaction can be performed for 1 hour at 37° C. with gentle shaking at300 rpm using short intervals. Spatial arrays are then washed with0.1×SSC buffer and treated with 60% DMSO at room temperature for 5minutes and then washed three times with 0.1×SSC buffer.

In order to detect the cDNA produced from the 18S rRNA, labeledoligonucleotide probes are generated that had sequence complementarilyto the 18S cDNA sequence. Oligonucleotides are identified that have alength between 18-23 nucleotides with an optimum at 20 nucleotides, amelting temperature (Tm) between 38-50° C. with the optimal temperatureat 42° C., and a content of guanine and cytosine of 30-60% with anoptimum at 50%. The first five bases of the 3′-ends are set to includetwo of either guanine or cytosine or one of each. Oligonucleotides arechecked for quality using Mfold (determination of secondary structure),Oligo Calc: Oligonucleotide Properties Calculator (determination ofself-dimerization and hairpin formation), and BLAST (determination ofoff-target binding). The sequence locations are picked for compatibilitywith both human (NR_003286.2) and mouse (NR_003278.3) 18S rRNA. Four ofthe oligonucleotide probes selected include: probe 1 (P1; SEQ ID NO: 4)GAGGAATTCCCAGTAAGT, probe 2 (P2; SEQ ID NO: 5) GAGATTGAGCAATAACAG, probe3 (P3; SEQ ID NO: 6) GTAGTTCCGACCATAAAC, and probe 4 (P4; SEQ ID NO: 7)GGTGACTCTAGATAACCT. Control oligonucleotide probes can be designed toinclude complementary sequences of three detection probes at a time witha 20 bases spacer sequence between each probe. The selectedoligonucleotide probes can be then designed to incorporate a Cy3fluorophore.

Next, labeled oligonucleotide probes are hybridized to the spatial arraycontaining cDNA produced from the 18S rRNA or containing control captureprobes. This step can include at least 4 successive rounds ofhybridization and imaging, with at least one round for each of the fourlabeled oligonucleotide probes. Following each round of hybridizationand imaging, the spatial array can be washed to remove the hybridizedprobe before continuing with a subsequent round of hybridization andimaging.

Hybridization of labeled oligonucleotide probes includes adding to thespatial arrays a pre-heated (e.g., heated to 50° C.) hybridizationmixture (10 mM Tris-HCl, 1 mM EDTA, 50 mM

NaCl, and 0.5 μM of fluorescently labelled probe) containing at least0.5 μM of one of the fluorescently labeled oligonucleotide probes (e.g.,one of P1, P2, P3 or P4). The spatial array can be then imaged using aDNA microarray scanner with the following settings: excitationwavelength 532 nm set to gain 70 and 635 nm set to 1. Following imaging,the spatial array is incubated with 60% DMSO at room temperature for 5minutes and washed three times with 0.1×SSC buffer to remove thehybridized probe. Subsequent rounds of hybridization and imaging areperformed with a different labeled oligonucleotide probe used in eachround (e.g., round 2 used P2, round 3 used P3, and round 4 used P4). Aninitial, pre-hybridization round (P0) of imaging are performed in orderto assess background fluorescence.

One image is generated per labeled oligonucleotide probe (P1-P4) andalso one where no fluorescently labeled probes were hybridized (P0).Normalization of Fluorescence Units (FU) data is done by subtraction ofthe auto-fluorescence recorded with PO and division with P1. Afteraligning the five images for a particular location in the tissue (oneimage from each probe, P1-P4, and one image from the location withoutlabeling), the images are loaded into a script and run in RStudio. TheScript can generate two different plots, one heat-map of RIN values andone image alignment error plot.

High quality RNA is defined as full-length (or close to full-length)transcripts, whereas low quality RNA is defined as fragmentedtranscripts. RIN values range from 1 to 10, with higher numbersindicating higher quality (e.g., less degraded, less fragmented) RNAsamples.

Spatial Assay for Transposase-Accessible Chromatin compositions

Provided herein are compositions for identifying the location of ananalyte in a biological sample. In some embodiments, a nucleotidemolecule composition including a) a spatial barcode b) a unique moleculec) capture domain; d) a functional domain; and e) a splintoligonucleotide. In some embodiments, a partially double-strandednucleotide molecule composition including: a) a spatial barcode; b) aunique molecular identifier; c) a capture domain; d) a functionaldomain; and e) a splint oligonucleotide. In some embodiments, acomposition including a) a capture probe including i) a spatial barcode;ii) a unique molecular identifier; iii) a capture domain; iv) afunctional domain; and v) a splint oligonucleotide and b) fragmentedgenomic DNA including i) a first adapter sequence comprising atransposon end sequence and a sequence complementary to the capturedomain; and ii) a second adapter sequence comprising the transposon endsequence and a second adapter sequence. In some embodiments, acomposition including a) a transposase enzyme monomer complexed with afirst adapter including i) a transposon end sequence; and ii) a sequencecomplementary to the capture domain; and b) a transposase enzyme secondmonomer complexed with a second adapter including i) a transposon endsequence; and ii) a second adapter sequence; c) genomic DNA; and d) acapture probe, including i) a spatial barcode; ii) a unique molecularidentifier; iii) a capture domain; iv) a functional domain; and v) asplint oligonucleotide. In some embodiments, a composition including a)a transposase enzyme monomer complexed with a first adapter including i)a transposon end sequence; ii) a sequence complementary to the capturedomain; wherein the 5′ end of the first adapter is phosphorylated and b)a transposase enzyme second monomer complexed with a second adapterincluding i) the transposon end sequence; ii) a second adapter sequence;wherein the 5′ end of the second adapter is phosphorylated; c) genomicDNA; and d) a capture probe including i) a spatial barcode; ii) a uniquemolecular identifier; iii) a capture domain; iv) a functional domain;and v) a splint oligonucleotide. In some embodiments, a compositionincluding a) a transposase enzyme dimer including i) a transposaseenzyme monomer complexed with a first adapter including 1) a transposonend sequence, 2) a sequence complementary to a capture domain; ii) atransposase enzyme second monomer complexed with a second adapterincluding 1) the transposon end sequence; 2) a second adapter sequence;b) genomic DNA; c) a capture probe, including i) a spatial barcode; ii)a unique molecular identifier; iii) a capture domain; iv) a functionaldomain; and v) a splint oligonucleotide. In some embodiments, acomposition including a) a transposase enzyme dimer including i) atransposase enzyme monomer complexed with a first adapter including 1) atransposon end sequence, 2) a sequence complementary to a capture domainwherein the 5′ end of the first adapter is phosphorylated; ii) atransposase enzyme second monomer complexed with a second adapterincluding 1) the transposon end sequence; 2) a second adapter sequencewherein the 5′ end of the second adapter is phosphorylated; c) genomicDNA; and d) a capture probe, including i) a spatial barcode; ii) aunique molecular identifier; iii) a capture domain; iv) a functionaldomain; and v) a splint oligonucleotide.

Spatial Transcriptomics

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample such as a tissue section) that include: (a) providinga biological sample on a substrate; (b) staining the biological sampleon the substrate, imaging the stained biological sample, and selectingthe biological sample or subsection of the biological sample to subjectto spatial analysis; (c) providing an array comprising one or morepluralities of capture probes on a substrate; (d) contacting thebiological sample with the array, thereby allowing a capture probe ofthe one or more pluralities of capture probes to capture the biologicalanalyte of interest; and (e) analyzing the captured biological analyte,thereby spatially detecting the biological analyte of interest. Anyvariety of staining and imaging techniques as described herein or knownin the art can be used in accordance with methods described herein. Insome embodiments, the staining includes optical labels as describedherein, including, but not limited to, fluorescent, radioactive,chemiluminescent, calorimetric, or colorimetric detectable labels. Insome embodiments, the staining includes a fluorescent antibody directedto a target analyte (e.g., cell surface or intracellular proteins) inthe biological sample. In some embodiments, the staining includes animmunohistochemistry stain directed to a target analyte (e.g., cellsurface or intracellular proteins) in the biological sample. In someembodiments, the staining includes a chemical stain such as hematoxylinand eosin (H&E) or periodic acid-schiff (PAS). In some embodiments,significant time (e.g., days, months, or years) can elapse betweenstaining and/or imaging the biological sample and performing spatialtranscriptomic analysis. In some embodiments, reagents for performingspatial analysis are added to the biological sample before,contemporaneously with, or after the array is contacted to thebiological sample. In some embodiments, step (d) includes placing thearray onto the biological sample. In some embodiments, the array is aflexible array where the plurality of spatially-barcoded features (e.g.,capture probes) are attached to a flexible substrate. In someembodiments, measures are taken to slow down a reaction (e.g., coolingthe temperature of the biological sample or using enzymes thatpreferentially perform their primary function at lower or highertemperature as compared to their optimal functional temperature) beforethe array is contacted with the biological sample. In some embodiments,step (e) is performed without bringing the biological sample out ofcontact with the array. In some embodiments, step (e) is performed afterthe biological sample is no longer in contact with the array. In someembodiments, the biological sample is tagged with an analyte captureagent before, contemporaneously with, or after staining and/or imagingof the biological sample. In such cases, significant time (e.g., days,months, or years) can elapse between staining and/or imaging andperforming spatial analysis. In some embodiments, the array is adaptedto facilitate biological analyte migration from the stained and/orimaged biological sample onto the array (e.g., using any of thematerials or methods described herein). In some embodiments, abiological sample is permeabilized before being contacted with an array.In some embodiments, the rate of permeabilization is slowed prior tocontacting a biological sample with an array (e.g., to limit diffusionof analytes away from their original locations in the biologicalsample). In some embodiments, modulating the rate of permeabilization(e.g., modulating the activity of a permeabilization reagent) can occurby modulating a condition that the biological sample is exposed to(e.g., modulating temperature, pH, and/or light). In some embodiments,modulating the rate of permeabilization includes use of external stimuli(e.g., small molecules, enzymes, and/or activating reagents) to modulatethe rate of permeabilization. For example, a permeabilization reagentcan be provided to a biological sample prior to contact with an array,which permeabilization reagent is inactive until a condition (e.g.,temperature, pH, and/or light) is changed or an external stimulus (e.g.,a small molecule, an enzyme, and/or an activating reagent) is provided.

Spatially-Resolved Gene Expression and Clustering in Invasive DuctalCarcinoma

The spatial gene expression of invasive ductal carcinoma tissue from afemale patient (ER+, PR−, HER2+) was profiled (BioIVT: Asterand—Case ID66320; Specimen ID 116899F). As a control, the healthy tissue sectionsadjacent to the tumor were obtained. 4 replicates were used for eachtissue type.

Spatially-resolved gene expression and clustering in invasive ductalcarcinoma reveal intra-tumor heterogeneity is shown in FIGS. 23A-H. FIG.23A shows a histological section of an invasive ductal carcinomaannotated by a pathologist. The section contains a large proportion ofinvasive carcinoma (22.344 mm² portion indicated by thick black line(outlined in black), three separate ductal cancer in situ regions(portions indicated by medium thickness black line 1.329 mm², 1.242 mm²,and 0.192 mm² (outlined in green in color figure)), and fibrous tissue.FIG. 23B shows a tissue plot with spots colored by unsupervisedclustering of transcripts. FIG. 23C shows a t-SNE plot of spots coloredby unsupervised clustering of transcripts. FIG. 23D shows a geneexpression heat map of the most variable genes between the 9 identifiedclusters. The region defined as fibrous tissue mostly corresponds toclusters 1, 7, and 8. Interestingly, a large region annotated asinvasive carcinoma by a pathologist contained spatial spots that wereassigned to DCIS (cluster 5). In addition, four subtypes of invasivecarcinoma with distinct molecular properties (clusters 2, 3, 4, and 6)were identified, revealing intra-tumor heterogeneity.

The expression levels of genes corresponding to human epidermal growthfactor receptor 2 (Her2 or ERBB2), estrogen receptor (ER or ESR1), andprogesterone receptor (PGR) in the tissue section are shown in FIG. 23E.It is clearly visible that ERBB2 and ESR1 are highly expressed in theinvasive carcinoma and DCIS regions while the expression of PR isabsent, consistent with the patient's diagnosis. One of the topdifferentially expressed genes from each cluster in the invasivecarcinoma region was selected (rectangular boxes in FIG. 23D), and itsexpression levels are located in the tissue as shown in FIG. 23F andoverlapped in one plot as shown in FIG. 23G. With the exception of PGR,all of these genes were highly up-regulated in the carcinoma tissuecompared to the adjacent normal tissue (FIG. 23H). Analysis revealedthat all of these up-regulated genes have implication in cancerprogression. Interestingly, in the subset of cluster 3, a longnon-coding RNA, of which abnormal expression has recently beenimplicated in tumor development (see, e.g., Zhang T, et al. LongNon-Coding RNA and Breast Cancer. Technol Cancer Res Treat. 2019,18,1533033819843889, incorporated herein by reference in its entirety),is one of the top differentially expressed genes. In glioblastoma,LINC00645 promotes epithelial-to-mesenchymal transition by inducingTGF-β (see, e.g., Li, C. et al. Long non-coding RNA linc00645 promotesTGF-β-induced epithelial-mesenchymal transition by regulatingmiR-205-3p-ZEB1 axis in glioma. Cell Death & Dis. 2019, 10, 272,incorporated herein by reference in its entirety).

During breast cancer progression, the myoepithelial cells, whichcontinue to surround preinvasive in situ carcinoma, gradually disappear(see, e.g., Gudjonsson, T. et al. Myoepithelial Cells: Their Origin andFunction in Breast Morphogenesis and Neoplasia. J. Mammary Gland Biol.Neoplasia. 2009, 10, 261, incorporated herein by reference in itsentirety). This phenomenon is clearly visualized in FIGS. 23I and 23Jwhere KRT14 (a gene signature of myoepithelial cells) was highlyexpressed around the lining of the duct in the normal tissue while itwas disappearing in the DCIS region in IDC tissue (FIG. 23I). Theextracellular matrix genes such as COL1A1 and FN1, key genes associatedwith invasion and metastasis, were highly upregulated while smoothmuscles and basal keratin were down-regulated in IDC (FIG. 23J).

Sequence Listing Synthetic PURAMATRIX ® polypeptide sequenceSEQ ID NO: 1 RADARADARADARADA Synthetic EAK16 polypeptide sequenceSEQ ID NO: 2 AEAEAKAKAEAEAKAK Synthetic KLD12 polypeptide sequenceSEQ ID NO: 3 KLDLKLDLKLDL 18s cDNA Probe 1 (P1)SEQ ID NO: 4 GAGGAATTCCCAGTAAGT 18s cDNA Probe 2 (P2)SEQ ID NO: 5 GAGATTGAGCAATAACAG 18s cDNA Probe 3 (P3)SEQ ID NO: 6 GTAGTTCCGACCATAAAC 18s cDNA Probe 4 (P4)SEQ ID NO: 7 GGTGACTCTAGATAACCT

VI. Spatial Cell-Based Analytical Methodology for Various BiologicalSamples

(a) Biological Samples and Analytes

It has been identified that any of the methods described herein can beused for various types of biological samples. Further, profiles ofbiological samples can be compared to profiles of other cells, e.g.,“normal,” or “healthy,” biological samples. Thus, in some embodiments ofany the methods described herein, the methods can provide for diagnosisof a disease. In some embodiments of any the methods described herein,the methods can be used in drug screening.

In some embodiments, the biological sample is an organism. In someembodiments, the biological sample is an organoid. In some embodiments,the biological sample is one or more cells from a cell culture (e.g.,suspended cells or adherent cells). In some instances, the biologicalsample is a plurality of live cell (e.g., from a cell culture (e.g.,suspended cells or adherent cells)).

Accordingly, provided herein are methods for profiling a biologicalanalyte, e.g., any of the analytes as described herein, in a biologicalsample such as an organism, an organoid, or an in vitro cell culture. Insome embodiments, these methods can include releasing the biologicalanalyte from one or more cells in an organism, an organoid, or an invitro cell culture. The released biological analyte can be bound by acapture probe as described herein at a distinct spatial position on asubstrate and detected. The bound biological analyte can then becorrelated with a barcode of the capture probe at a distinct spatialposition of the substrate. Such methods can be useful for correlating abiological analyte to a spatial location in a cell culture, an organism,or an organoid on an array as described herein. For example, in someembodiments, provided herein are methods for identifying a location of abiological analyte present in a biological sample, wherein thebiological sample is an organism, an organoid, or an in vitro cellculture, the method comprising: (a) contacting the biological samplewith a substrate comprising a plurality of capture probes, wherein acapture probe of the plurality of capture probes comprises a spatialbarcode and a capture domain that binds specifically to a biologicalanalyte present in the biological sample; and (b) determining (i) all ora part of the sequence of the biological analyte specifically bound tothe capture domain, or a complement thereof, and (ii) all or a part ofthe sequence of the spatial barcode or a complement thereof, and usingthe determined sequences of (i) and (ii) to identify the location of thebiological analyte in the biological sample. In some embodiments,provided herein are methods of identifying a location of a biologicalanalyte in a biological sample, wherein the biological sample is anorganism, an organoid, or an in vitro cell culture, the methodcomprising: (a) contacting a plurality of analyte capture agents to abiological sample disposed on a substrate, wherein: an analyte captureagent of the plurality of the analyte capture agents comprises (i) ananalyte binding moiety that binds specifically to the biologicalanalyte, (ii) an analyte binding moiety barcode; and (iii) an analytecapture sequence; and the substrate comprises a plurality of captureprobes, wherein a capture probe of the plurality comprises a spatialbarcode and a capture domain that binds specifically to the analytecapture sequence; (b) determining the sequence of (i) all or a portionof the spatial barcode or a complement thereof, and (ii) all or aportion of the analyte binding moiety barcode, or a complement thereof,and using the determined sequences of (i) and (ii) to identify thelocation of the biological analyte in the biological sample. In someembodiments, the biological sample is a cell culture (e.g., a suspendedor adherent cell as described herein), an organism, or an organoid.

In some embodiments, the biological sample is derived from a homogenouscellular population. In some embodiments, the biological sample isderived from a heterogeneous cellular population. In some embodiments,the heterogeneous cellular population is a culture of cells, a wholeorganism, or an organoid).

In some embodiments, the biological sample is an organism. In someembodiments, the organism is a nematode (e.g., Caenorhabditis elegans),Danio rerio (zebrafish), Xenopus tropicalis, Xenopus laevis, Daphniaspp., Coelopidae, Drosophila, Dipsidae, Branchiostoma floridae, Galleriamellonella, Macrostomum lignano, Oscarella carmela, Pristionchuspacificus, Schmidtea mediterranea, Symsagittifera roscoffensis,Trichoplax adhaerens, Tubifex tubifex, Nothobranchius furzeri, orPoecilia reticulate.

In some embodiments, the biological sample is obtained from a prokaryotesuch as a bacterium, e.g., Escherichia coli, Staphylococci, orMycoplasma pneumoniae; or an archaea. In some embodiments, thebiological sample is obtained from a virus. For example, the biologicalsample can be obtained from a virus such as Hepatitis C virus or humanimmunodeficiency virus. In some embodiments, the biological sample isobtained from a viroid.

In some embodiments, the biological sample is an organoid. As referredto herein an “organoid” is a self-organized, three-dimensional tissueculture derived from stem cells. An organoid can be a miniaturized andsimplified version of an organ that shows realistic micro-anatomy.Non-limiting examples of an organoid include a cerebral organoid, anintestinal organoid, a stomach organoid, a lingual organoid, a thyroidorganoid, a thymic organoid, a testicular organoid, a hepatic organoid,a pancreatic organoid, an epithelial organoid, a lung organoid, a kidneyorganoid, a gastruloid, a cardiac organoid, and a retinal organoid.

In some embodiments, the biological sample is a cell culture. A cellculture as described herein can comprise a plurality of cells. In someembodiments, the plurality of cells is from a cell culture of suspendedcells or a cell culture of adherent cells as described herein. Forexample, the adherent cells can be from cell lines comprising at leastone of BT549, HS 578T, MCF7, MDA-MB-231, MDAMB-468, T-47D, SF268, SF295,SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29,KM12, SW620, 786-O, A498, ACHN, CAKI, RXF 393, SN12C, TK-10, UO-31,A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, LOXIMVI, M14, MALME-3M, MDA-MB-435, SK-, EL-2, SK-MEL-28, SK-MEL-5,UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3,NCI-ADR-RES, DU145, PC-3, DU145, H295R, HeLa, KBM-7, LNCaP, MCF-7,MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D, THP-1, U87, vero, MC3T3, GH3,PC12, dog MDCK kidney epithelial, Xenopus A6 kidney epithelial,zebrafish AB9, or Sf9 insect epithelial cell lines. In some embodiments,the biological sample is a plurality of cells from a disassociatedtissue or a tissue section. In some embodiments, the biological sampleis a plant. In some embodiments, the plant is a monocotyledonous plant(i.e., a monocot). For example, a monocotyledonous plant includes but isnot limited to corn, rice, sugarcane, switchgrass, meadow grass species,Chinese sorghum, barley, wheat and oat, and durum wheat. In someembodiments, the plant is a dicotyledonous plant (i.e., a dicot). Forexample, a dicotyledonous plant includes but is not limited to soybean,cotton, rape, flax, sunflower, legume, clover, peanut and tomato.

In some embodiments, the biological samples described herein includeanalytes of interest. In some embodiments, one or more biologicalanalytes are detected and analyzed in a biological sample. In someembodiments, the analyte is any analyte as described herein, includingbut not limited to a nucleic acid, a lipid, a carbohydrate, or aprotein. In some embodiments, the analyte is an organelle as describedherein.

In some embodiments, the analyte can be a cell surface marker. In someembodiments, a plurality of analyte capture agents can be disposed onthe surface of a substrate as described herein. In some embodiments, theplurality of analyte capture agents to a biological sample can be anantibody. In some embodiments, the plurality of analyte capture agentsto a biological sample can bind to a cell surface marker. Non-limitingexamples of a cell surface marker include CD45, CD3, CD4, CD8, CD56,CD19, CD20, CD11c, CD14, CD33, CD66b, CD34, CD41, CD61, CD235a, CD146,and epithelial cellular adhesion molecule (EpCAM). An analyte captureagent disposed on the surface of a substrate can be used to bind a cellthat has the corresponding cell surface maker.

In some embodiments, the biological sample is contacted with a substratewhen the biological sample is in a bulk solution. In some embodiments,the bulk solution permeabilizes one or more membranes of a biologicalsample, allowing access to a biological analyte. A bulk solution can beany bulk solution as described herein.

Tissues and cells can be obtained from any source. For example, tissuesand cells can be obtained from single-cell or multicellular organisms(e.g., a mammal). Tissues and cells obtained from a mammal, e.g., ahuman, often have varied analyte levels (e.g., gene and/or proteinexpression) which can result in differences in cell morphology and/orfunction. The position of a cell or a subset of cells (e.g., neighboringcells and/or non-neighboring cells) within a tissue can affect, e.g.,the cell's fate, behavior, morphology, and signaling and cross-talk withother cells in the tissue. Information regarding the differences inanalyte levels (gene and/or protein expression) within different cellsin a tissue of a mammal can also help researchers identify and elucidateanalyte levels in the single-cell or multicellular organisms (e.g., amammal) based on the detected differences in analyte levels withindifferent cells in the tissue. Differences in analyte levels withindifferent cells in a tissue of a mammal can also provide information onhow tissues (e.g., healthy and diseased tissues) function and/ordevelop. Differences in analyte levels within different cells in atissue of a mammal can also provide information of different mechanismsof disease pathogenesis in a tissue and mechanism of action of atherapeutic treatment within a tissue. Differences in analyte levelswithin different cells in a tissue of a mammal can also provideinformation on drug resistance mechanisms and the development of thesame in a tissue of a mammal. Differences in the presence or absence ofanalytes within different cells in a tissue of a multicellular organism(e.g., a mammal) can provide information on drug resistance mechanismsand the development of the same in a tissue of a multicellular organism.

(b) Substrate(s) for Cell-Based Analysis

One or more substrates can be used in the methods as described herein.In some embodiments, a biological sample can be disposed upon asubstrate comprising a plurality of capture probes as described herein.In some embodiments, wherein one substrate is used, a biological sampleis disposed upon a substrate comprising a plurality of capture probes asdescribed herein (i.e., onto the same substrate). See, for example, FIG.26A.

In some embodiments, a biological sample can be disposed upon a firstsubstrate and contacted with a second substrate comprising a pluralityof capture probes. In some embodiments, wherein two substrates are used,a biological sample is disposed upon a first substrate, and the firstsubstrate with the biological sample is contacted with a secondsubstrate that comprises a plurality of capture probes as describedherein. See, for example, FIG. 26B. As another example, a plurality ofcells, e.g., from a cell culture, can be distributed upon either a firstsubstrate or a second substrate. In some embodiments, wherein the firstsubstrate comprises a plurality of capture probes, a cell of theplurality of cells is distributed onto the first substrate such that thecell occupies a distinct feature on the first substrate. In someembodiments, a cell of the plurality of cells is distributed onto afirst substrate and contacted with a second substrate comprising aplurality of capture probes. In other words, the biological sample(e.g., cells) are one one slide and the plurality of capture probes areon another slide.

In some embodiments, the first substrate is contacted with thebiological sample. In some embodiments, the first substrate is contactedwith the second substrate. In some embodiments, the first substrate iscontacted with the second substrate prior to releasing the biologicalanalyte from the biological sample. In some embodiments, the biologicalsample is lysed or permeabilized, allowing capture probes to associatewith (e.g., hybridize to) a target analyte. In some embodiments, afterassociation of the capture probe with an analyte, further steps,including cDNA synthesis and purification, and sequencing are performedusing methods described herein.

In some embodiments, a plurality of cells (e.g., a cell culturecomprising a plurality of cells) is distributed onto the substrate(e.g., the first substrate). In some embodiments, wherein the substrate(e.g., the first substrate) comprises a plurality of features, a cell ofthe plurality of cells is distributed onto the substrate such that thecell occupies a distinct feature on the substrate. In some embodiments,wherein the substrate (e.g., the first substrate) comprises a pluralityof features, members of the plurality of cells are distributed over thesubstrate (e.g., the first substrate) until about all of the features onthe substrate are bound to a cell. In some embodiments, wherein thefirst substrate comprises a plurality of features, the features aresmaller than the minimum cell diameter. In some embodiments, wherein thefirst substrate comprises a plurality of features, cells not bound to afeature on the substrate are washed off. In some embodiments, cells notbound to a feature on the substrate are washed off prior to releasingthe biological analyte. In some embodiments, members of the plurality ofcells are distributed over the substrate prior to the binding of membersof the plurality of cells to members of the plurality of captureantibodies.

In some embodiments, the surface of the substrate (e.g., the firstsubstrate) is coated with a cell-permissive coating, e.g., acell-permissive coating as described herein. In some embodiments, thecell-permissive coating includes at least one of a biological materialand/or a synthetic material. In some embodiments, the cell-permissivecoating includes coatings that feature one or more extracellular matrix(ECM) components (e.g., proteoglycans and fibrous proteins such ascollagen, elastin, fibronectin and laminin), poly-lysine,poly(L)-ornithine, and/or a biocompatible silicone (e.g., CYTOSOFT®).For example, a cell-permissive coating that includes one or moreextracellular matrix components can include collagen Type I, collagenType II, collagen Type IV, elastin, fibronectin, laminin, and/orvitronectin. In some embodiments, the cell-permissive coating includes asolubilized basement membrane preparation extracted from theEngelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., MATRIGEL®). In someembodiments, the cell-permissive coating includes collagen.

In some embodiments, a biological sample can be immobilized on thesubstrate. For example, at least one cell of a plurality of cells can beimmobilized on the substrate after the at least one cell is distributedonto the substrate. In some embodiments, the biological sample isimmobilized on the substrate prior to releasing the biological analyte.In some embodiments, the biological sample is immobilized by adding ahydrogel. In some embodiments, the biological sample is immobilized byapplying an electric field.

In some embodiments, before the biological sample is distributed onto asubstrate, it is permeabilized to allow access to one or more biologicalanalytes. In some embodiments, after the biological sample isdistributed onto a substrate, it is permeabilized to allow access to oneor more biological analytes. Permeabilization can be performed using anyreagents and techniques described herein. In some embodiments, thereleasing the biological analyte comprises permeabilizing the biologicalsample, e.g., using any of the permeabilization methods as describedherein. In some embodiments, a method as described herein furthercomprises fixing the biological sample prior to permeabilization.

In some embodiments, the methods further comprise imaging the biologicalsample, e.g., imaging using any of the methods as described herein.

VII. Cell Migration Assays and Spatial Cell-Based Analytical Methodology

In some embodiments, methods as described herein can be combined with acell migration assay. For example, cells can be disposed on an array asdescribed herein and allowed to migrate (FIGS. 29A and 29B). In someembodiments, the substrate is spotted with barcoded probes and cellcapture molecules (FIG. 29A, middle). In some embodiments, the substrateis spotted with barcoded probes, cell capture molecules, and one or morecells (FIG. 29A, right). In some embodiments, the substrate includes oneor more capture probes and/or one or more capture antibodies. See FIG.29B. In some embodiments, the one or more capture antibodies arespecific for one or more cell surface markers. In some embodiments, thecell migration distance can be measured and correlated to a biologicalanalyte. In some embodiments, the substrate includes a plurality ofcapture probes that provide information (e.g., a spatial barcode) thatprovide a specific position on the substrate. In some embodiments, onecan use the spatial barcode information to determine the location of themigrated cell. For example, in some embodiments, a method for profilingat least one biological analyte present in a cell comprises: (a)allowing a cell of a plurality of cells to migrate on a substratecomprising a plurality of capture probes, wherein a capture probecomprises a spatial barcode and a capture domain; (b) releasing abiological analyte from the cell such that the biological analyte isbound by a capture probe at a distinct spatial position of thesubstrate; (c) detecting the bound biological analyte; and (d)correlating the biological analyte with a molecular barcode of thecapture probe at the distinct spatial position of the substrate, thusprofiling at least one biological analyte as having been present in acell at a distinct spatial position.

In some embodiments, presence of analytes can be analyzed in a settingof cell migration in one or more particular cell(s). For example, insome embodiments, presence of analytes during migration of cancer cellscan be detected. For example, in some embodiments, presence of analytesduring migration of inflammatory cells can be detected.

A substrate used in a cell migration assay can comprise one or morechannels that are disposed on the substrate (FIG. 28), e.g., to confinecell migration to one dimension along the substrate. Additionally, thechannels can direct the migration of a cell such that it does notcontact another cell on the substrate (e.g., the channels do not overlapwith each other). In some embodiments, the one or more channels confinecell migration along one axis of the substrate. In some embodiments, theone or more channels confine cell migration along more than one axis ofthe substrate. In some embodiments, the one or more channels confinecell migration along two axes of the substrate. In some embodiments, achannel does not overlap with any other channel. In some embodiments,the channels are wide enough to accommodate a cell (e.g., a channel canhave a width of about 2 μm to about 10 μm). For example, a channel canhave a width of about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm.

In some embodiments, cells can be disposed on a substrate as describedherein and allowed to migrate. Cell migration in cell migration assayscan be used to measure target phenotypes (e.g., phenotype forinvasiveness). In some embodiments, the cell migration distance can bemeasured and correlated to a biological analyte. Reagents can be addedto the substrate to facilitate cell migration. For example, thesubstrate can be coated with one or more extracellular matrix (ECM)components (e.g., basement membrane extract (BME), laminin I, collagenI, collagen IV, fibronectin, vitronectin, elastin), a cell culturemedium, a chemoattractant (i.e., a substance which attracts motile cellsof a particular type), a chemorepellant, or a combination thereof.Chemoattractants and chemorepellents are inorganic or organic substancespossessing chemotaxis-inducer effect in migrating cells. In someembodiments, a chemoattractant is a positive chemoattractant; that is,it promotes movement toward a higher concentration of thechemoattractant (e.g., molecule or chemical). In some embodiments, achemoattractant is a negative chemoattractant; that is, it promotesmovement against a higher concentration of the chemoattractant (e.g.,molecule or chemical). Chemoattractants are discussed in Halova et al.,Front Immunol. 2012; 3: 119, which is incorporated by reference in itsentirety. For example, chemoattractants include but are not limited to astem cell factor (SCF) recognized by c-Kit, antigen, which binds toimmunoglobulin E (IgE) anchored to the high affinity IgE receptor(FcεRI); highly cytokinergic (HC) IgE recognized by FcεRI; lipidmediator sphingosine-1-phosphate (S1P), which binds to G protein-coupledreceptors (GPCRs); eicosanoids [prostaglandin E2 and D2, leukotriene(LT) B4, LTD4, and LTC4, and others] and chemokines (CC, CXC, C, andCX3C), which also bind to various GPCRs; isoforms of transforming growthfactor (TGF) β1-3, which are sensitively recognized by TGF-βserine/threonine type I and II β receptors; adenosine; C1q, C3a, and C5acomponents of the complement; 5-hydroxytryptamine; neuroendocrinepeptide catestatin; and tumor necrosis factor-α.

In some embodiments, a reagent such as a chemoattractant orchemorepellant can be disposed on only a portion of the substrate,present as a gradient along the one or more axis or channels of thesubstrate, or a combination thereof. In some embodiments, achemoattractant or chemorepellant is present at only one end of the oneor more channels. In some embodiments, the chemoattractant orchemorepellant is bound to one or more ECM components.

In some embodiments, the method can further comprise imaging themigration patterns of one or more cells in a biological sample (e.g.,organism, organoid, cell culture) on the substrate as described herein.Imaging can be used to determine the spatial location of a cell on thesubstrate, e.g., the spatial location in a channel on the substrate,and/or to determine the distance of migration of a cell. For example,imaging can be performed before and/or after cell migration. In someembodiments, imaging is performed prior to releasing the biologicalanalyte.

In some embodiments, detection of the biological analyte is correlatedwith the cell migration distance. For example, an analyte can bedetected before, during, and/or after migration that providesinformation about the distance traveled. In some embodiments, detectionof an analyte is correlated with the cell migration distance of at leastabout 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm,about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 15 μm, about 20μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm,about 50 μm, about 75 μm, about 100 μm, about 150 μm, about 200 μm,about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm,about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm,about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm,about 1 mm, or more.

VIII. Spatial Analytical Methodology and Selective Release of aBiological Analyte

(a) Methods for Profiling a Selectively-Released Biological Analyte in aBiological Sample

Provided herein are methods for profiling a biological analyte, e.g.,any of the analytes as described herein, in a biological sample thatinclude selectively releasing the biological analyte. In someembodiments, these methods can include selectively releasing thebiological analyte from one or more of: a subcellular region of a cellin a biological sample, a particular type of cell in a biologicalsample, and a region of interest of a biological sample. The releasedbiological analyte can be bound by a capture probe as described hereinat a distinct spatial position on a substrate and detected. The boundbiological analyte can then be correlated with a barcode of the captureprobe at a distinct spatial position of the substrate. Such methods canbe useful for correlating a subcellular region of a cell, a particulartype of cell, and/or a region of interest of a biological sample to aspatial location for a biological analyte.

Biological samples can be processed to selectively release a biologicalanalyte from a subcellular region of a cell, a particular type of cell,and/or a region of interest of a biological sample according toestablished methods. For example, a biological analyte can beselectively released from a region of interest using any of the methodsas described herein (e.g., microsurgery, laser capture microdissection,chunking, a microtome, dicing, trypsinization, labelling, and/orfluorescence-assisted cell sorting). In some embodiments, a biologicalanalyte can be selectively released from a subcellular region of a cell,a particular type of cell, and/or a region of interest of a biologicalsample by selective permeabilization.

In some embodiments, “selective permeabilization” can refer to apermeabilization method that can permeabilize a membrane of asubcellular region while leaving a different subcellular regionsubstantially intact (e.g., biological analytes are not released fromsubcellular region due to the applied permeabilization method).Non-limiting examples of selective permeabilization methods used topermeabilize a membrane of a subcellular region while leaving adifferent subcellular region substantially intact include usingelectrophoresis and/or applying a permeabilization agent or a lysisagent. Several methods for selective permeabilization of a subcellularregion are known to one of skill in the art including the methodsdescribed in Lu et al. Lab Chip. 2005 January; 5(1):23-9; Niklas et al.Anal Biochem. 2011 Sep. 15; 416(2):218-27; Cox and Emili. Nat Protoc.2006; 1(4):1872-8; Chiang et al. J Biochem. Biophys. Methods. 2000 Nov.20; 46(1-2):53-68; and Yamauchi and Herr et al. Microsyst. Nanoeng.2017; 3. pii: 16079; each of which is incorporated herein by referencein its entirety. In some embodiments, “selective permeabilization” canrefer to the selective permeabilization of a specific cell type. Forexample, “selective permeabilization” can refer to permeabilizing onecell type while leaving a different cell type substantially intact(e.g., biological analytes are not released from the cell due to theapplied permeabilization method). A cell that is a “different cell type”than another cell can refer to a cell from a different taxonomickingdom, a prokaryotic cell versus a eukaryotic cell, a cell from adifferent tissue type, etc. Many methods are known to one of skill inthe art for selectively permeabilizing different cell types.Non-limiting examples include applying a permeabilization reagent,electroporation, and/or sonication. See, e.g., International ApplicationNo. WO 2012/168003; Han et al. Microsyst Nanoeng. 2019 Jun. 17; 5:30;Kashyap et al. Sci Rep. 2016; 6: 29579; Oren and Shai. Biochemistry.1997 Feb. 18; 36(7):1826-35; Algayer et al. Molecules. 2019 May 31;24(11). pii: E2079; Hipp et al. Leukemia. 2017 October; 31(10):2278;International Application No. WO 2012/168003; and U.S. Pat. No.7,785,869; all of which are incorporated by reference herein in theirentireties. In some embodiments, “selective permeabilization” can referto selectively permeabilizing a region of interest of a biologicalsample. For example, the region of interest can be selectivelyadministered a permeabilization or lysis agent. See also Gould et al.Oncotarget. 2018 Mar. 20; 9(21): 15606-15615. In some embodiments, thepermeabilization or lysis agent is administered by contacting thebiological sample with a hydrogel that comprises the permeabilization orlysis agent.

In some embodiments, a method for identifying a location of a biologicalanalyte present in a subcellular region of a cell in a biological samplecan include: (a) contacting a biological sample comprising a cell with asubstrate comprising a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises a spatial barcode anda capture domain; (b) releasing a biological analyte from a subcellularregion of the cell in, wherein the biological analyte is specificallybound by the capture probe; and (c) determining (i) all or a part of thesequence of the biological analyte specifically bound to the capturedomain, or a complement thereof, and (ii) all or a part of the sequenceof the spatial barcode or a complement thereof, and using the determinedsequences of (i) and (ii) to identify a location of the biologicalanalyte in the biological sample. In some embodiments, a method foridentifying a location of profiling a biological analyte present in asubcellular region of a cell in a biological sample can include: (a)contacting a plurality of analyte capture agents to a biological samplecomprising a cell disposed on a substrate, wherein: an analyte captureagent of the plurality of the analyte capture agents comprises (i) ananalyte binding moiety that binds specifically to the biologicalanalyte, (ii) an analyte binding moiety barcode; and (iii) an analytecapture sequence; and the substrate comprises a plurality of captureprobes, wherein a capture probe of the plurality of capture proteinscomprises (i) a spatial barcode, and (ii) a capture domain; (b)releasing a biological analyte from a subcellular region of the cell,wherein the analyte capture sequence is specifically bound by thecapture domain; and (c) determining the sequence of (i) all or a portionof the spatial barcode or a complement thereof, and (ii) all or aportion of the analyte binding moiety barcode, or a complement thereof,and using the determined sequences of (i) and (ii) to identify alocation of the biological analyte in the biological sample. In someembodiments, the biological analyte is released from the subcellularregion of the cell wherein release of other biological analytes fromother subcellular regions of the cell is substantially prevented. Insome embodiments, “substantially prevented” refers to less than 50%,less than 45%, less than 40%, less than 35%, less than 30%, less than25%, less than 20%, less than 18%, less than 16%, less than 14%, lessthan 12%, less than 10%, less than 8%, less than 6%, less than 5%, lessthan 4%, less than 3%, less than 2%, or less than 1% of other biologicalanalytes being released from other subcellular regions of the cell. Insome embodiments, the biological analyte is released from thesubcellular region of the cell wherein release of other biologicalanalytes from other subcellular regions of the cell is completelyprevented. As used herein, a “subcellular region” can refer to anysubcellular region. For example, a subcellular region can refer tocytosol, a mitochondria, a nucleus, a nucleolus, an endoplasmicreticulum, a lysosome, a vesicle, a Golgi apparatus, a plastid, avacuole, a ribosome, and cytoskeleton. In some embodiments, thesubcellular region comprises at least one of cytosol, a nucleus, amitochondria, and a microsome. In some embodiments, the subcellularregion is cytosol. In some embodiments, the subcellular region is anucleus. In some embodiments, the subcellular region is a mitochondria.In some embodiments, the subcellular region is a microsome.

In some embodiments, the subcellular region includes the cytosol. Insome embodiments, a cell membrane is permeabilized to extract one ormore components in a cell. See FIG. 30. In some embodiments, a cellmembrane is permeabilized to extract the cytosolic fraction and provideaccess to one or more analytes in the cytosolic fraction. In someembodiments, the subcellular region includes the one or more organelles.In some embodiments, the one or more organelles is permeabilized toextract the organelle fraction(s) and provide access to one or moreanalytes in the organelle fraction(s). In some embodiments, thesubcellular region includes the nucleus. In some embodiments, a nuclearmembrane is permeabilized to extract the nuclear fraction and provideaccess to one or more analytes in the nuclear fraction. See, forexample, FIG. 30. In some embodiments, the methods disclosed hereinutilize a bulk solution. See, for example, FIG. 31. In some embodiments,the biological sample is provided onto the substrate and immobilizedusing a bulk solution. Afterwards, downstream analysis as disclosedherein (e.g., capture transcripts, synthesize cDNA, collect barcodedcDNA for sequencing, capture nuclear fraction targets, collect barcodedtargets for sequencing) can be performed. In some embodiments, the cellmembrane in permeabilized to collect cytosolic fraction (e.g., for 3′transcriptomic assay). See, for example, FIG. 31, middle. In someembodiments, the nucleus is permeabilized for ATAC-seq treatment, thencollect targets from nuclear fraction. See, for example, FIG. 31,bottom.

In some embodiments, a method for profiling a biological analyte presentin a region of interest of a biological sample can include: (a)contacting a biological sample with a substrate comprising a pluralityof capture probes, e.g., capture probes as described herein, wherein acapture probe comprises a spatial barcode and a capture domain; (b)releasing a biological analyte from the particular cell type in thebiological sample, wherein the biological analyte is bound by thecapture probe at a distinct spatial position of the substrate; (c)detecting the bound biological analyte; and (d) correlating thebiological analyte with a barcode of the capture probe at the distinctspatial position of the substrate, thus profiling the biological analyteas having been present in the biological sample at a distinct spatialposition.

In some embodiments, a method for profiling a biological analyte presentin a region of interest of a biological sample can include: (a)contacting a biological sample with a substrate comprising a pluralityof capture probes, e.g., capture probes as described herein, wherein acapture probe comprises a spatial barcode and a capture domain; (b)releasing a biological analyte from a region of interest of thebiological sample, wherein the biological analyte is bound by thecapture probe at a distinct spatial position of the substrate; (c)detecting the bound biological analyte; and (d) correlating thebiological analyte with a barcode of the capture probe at the distinctspatial position of the substrate, thus profiling the biological analyteas having been present in the biological sample at a distinct spatialposition. In some examples, a region of interest can be permeabilized orlysed while areas outside the region of interest are not permeabilizedor lysed. For example, in some embodiments, a region of interest can becontacted with a hydrogel comprising a permeabilization or lysingreagent. In some embodiments, the area(s) outside the region of interestare not contacted with the hydrogel comprising the permeabilization orlysing reagent. In some embodiments, the area outside the region ofinterest of the biological sample is removed by microdissection.

In some embodiments, the methods further comprise imaging the biologicalsample, e.g., imaging using any of the methods as described herein.

(b) Methods for More than One Selective Release of a Biological Analytein a Biological Sample

In some embodiments, biological analytes can be released from abiological sample and bound to capture probes more than once (e.g., morethan one time before and after an event (e.g., before and aftertreatment of a biological sample)). In some embodiments, the methodsdisclosed herein include more than one selective permeabilization stepthat releases a biological analyte from a biological sample. In someembodiments, a first biological analyte from a subcellular region of acell, a particular type of cell, a region of interest of a biologicalsample, or a combination thereof, can be released from a biologicalsample and bound to capture probe. See e.g., FIG. 32A. In someembodiments, a first biological analyte from a biological sample isdetected by a capture probe (using the target capture sequence in thecapture probe). In some embodiments, the first biological analyte (e.g.,an RNA sequence) is reverse transcribed into cDNA. After, copies of thefirst biological analyte are removed, allowing for additional biologicalanalytes to hybridize or bind to the probe. See e.g., FIG. 32B. In someembodiments, one or more additional biological analytes (e.g., a secondbiological analyte, a third biological analyte, or a fourth biologicalanalyte) can be released from a different subcellular region of a cell,particular type of cell, region of interest of a biological sample, orcombination thereof, and binding of the one or more additionalbiological analytes to one or more capture probes.

In some embodiments, the biological sample is in a bulk solution.

In some embodiments, in between hybridization of a first biologicalanalyte and subsequent biological analytes, one or more probes can berepaired. In some embodiments, repair of a probe includes restoring theprobe to the original nucleic acid sequence and length prior to contactwith the first biological analyte using any methods of extending orretracting the size of a probe (e.g., a nucleic acid) as describedherein.

In some embodiments, a biological analyte from a subcellular region of acell, a particular type of cell, a region of interest of a biologicalsample, or a combination thereof, that is bound by a capture probe at adistinct spatial position of the substrate can be different from abiological analyte from any other subcellular region of the cell,particular type of cell, region of interest of the biological sample, orcombination thereof, that is bound by another capture probe at adistinct spatial position of the substrate. For example, a biologicalanalyte bound by a capture probe at a distinct spatial position of thesubstrate from a first subcellular region, first particular type ofcell, first region of interest, or combination thereof, can be differentfrom a biological analyte bound by a capture probe at a distinct spatialposition of the substrate from a second, third, and/or fourthsubcellular region, particular type of cell, region of interest, orcombination thereof. In some embodiments, a biological analyte bound bya capture probe at a distinct spatial position of the substrate from afirst subcellular region, first particular type of cell, first region ofinterest, or combination thereof, is the same as a biological analytebound by a capture probe at a distinct spatial position of the substratefrom any other subcellular region, particular type of cell, region ofinterest, or combination thereof, e.g., a second, third, fourth, orfifth subcellular region, particular type of cell, region of interest,or combination thereof. In some embodiments, wherein more than onebiological analyte from a subcellular region of the cell, particulartype of cell, region of interest of the biological sample, orcombination thereof, is bound by a capture probe at a distinct spatialposition of the substrate, as least one of the bound biological analytesis different from another bound biological analyte.

In some embodiments, a biological analyte can be released from more thanone subcellular region. For example, in some embodiments, the methodfurther includes: releasing a biological analyte from a subcellularregion, e.g., a second subcellular region, of a cell from the biologicalsample, wherein the biological analyte is bound by a capture probe at adistinct spatial position of the substrate; detecting the boundbiological analyte; and correlating the biological analyte with abarcode of the capture probe at the distinct spatial position of thesubstrate, wherein the second subcellular region is different than afirst subcellular region. In some embodiments, the detection steps arerepeated one (e.g., for a third subcellular region), two (e.g., for afourth subcellular region), three (e.g., for a fifth subcellularregion), or four (e.g., for a sixth subcellular region) times. In someembodiments, the subcellular region of a cell is cytosol and thesubcellular region of a cell includes at least one of a nucleus, amitochondria, or a microsome.

In some embodiments, a biological analyte can be released from more thanone particular type of cell. For example, in some embodiments, themethod further comprises: releasing a biological analyte from aparticular type of cell, e.g., a second particular type of cell, of acell from the biological sample, wherein the biological analyte is boundby a capture probe at a distinct spatial position of the substrate;detecting the bound biological analyte; and correlating the biologicalanalyte with a barcode of the capture probe at the distinct spatialposition of the substrate, wherein the particular type of second cell isdifferent than the particular type of a first cell. In some embodiments,the detection steps are repeated one (e.g., for a third particular typeof cell), two (e.g., for a fourth particular type of cell), three (e.g.,for a fifth particular type of cell), or four (e.g., for a sixthparticular type of cell) times.

In some embodiments, a biological analyte can be released from more thanone region of interest. For example, in some embodiments, the methodfurther comprises: releasing a biological analyte from a region ofinterest, e.g., a second region of interest, of a cell from thebiological sample, wherein the biological analyte is bound by a captureprobe at a distinct spatial position of the substrate; detecting thebound biological analyte; and correlating the biological analyte with abarcode of the capture probe at the distinct spatial position of thesubstrate, wherein the region of interest is different than the regionof interest of a first biological analyte. In some embodiments, thedetection steps are repeated one (e.g., for a third region of interest),two (e.g., for a fourth region of interest), three (e.g., for a fifthregion of interest), or four (e.g., for a sixth region of interest)times.

The method steps can be performed in various orders. Additionally, thesubstrate can be washed before or after each step. In some embodiments,wash steps removes unbound analytes.

There are multiple methods that can be used in detecting biologicalanalytes from a biological sample on which more than one release, e.g.,selective permeabilization, step is performed. Non-limiting examplesinclude using second strand synthesis, selective capture probe cleavage,and multiple substrates.

In some embodiments, second strand synthesis is used in the detection ofa biological analyte from a biological sample on which more than onerelease, e.g., selective permeabilization, step is performed. Forexample, a biological analyte (e.g., a first biological analyte) from afirst release step can be bound to a capture probe on a substrate andsecond strand synthesis, e.g., as described herein, can be performed toform a second strand comprising a sequence that corresponds to thesequence of at least a portion of the biological analyte (e.g., thefirst biological analyte). After the second strand from the firstrelease step is collected, a second release step can be performed tobind a biological analyte (e.g., a second biological analyte) to acapture probe, and second strand synthesis can be performed again toform a second strand comprising a sequence that corresponds to at leasta portion of the sequence of the biological analyte (e.g., the secondbiological analyte) and the second strand for the second release stepcan be collected. See, for example, FIGS. 32A-32B. The second strandsynthesis steps may be repeated one, two, three, four, or more times.The collected second strands from each release step can be detected andused to correlate the first, second, third, or fourth biological analytewith the barcode of the capture probe to which the biological analytewas bound at a distinct spatial position of the substrate.

In some embodiments, selective capture probe cleavage can be used in thedetection of a biological analyte from a biological sample on which morethan one release, e.g., selective permeabilization, step is performed.For example, a biological analyte (e.g., a first biological analyte)from a first release step can be bound to a first capture probe on asubstrate and a first extended capture probe can be generated (e.g.,cDNA synthesis can be performed on the first capture probe bound to thebiological analyte). The first extended capture probe can be selectivelycleaved (e.g., leaving any second, third, or fourth capture probesintact) and collected. After the first capture probe is collected, asecond release step can be performed to bind a biological analyte (e.g.,a second biological analyte) to a second capture probe on the substrateand a second extended capture probe can be generated (e.g., cDNAsynthesis can be performed on the second capture probe bound to thebiological analyte). The second extended capture probe can then becleaved and collected. See, for example, FIG. 33. The selective captureprobe cleavage steps may be repeated one, two, three, four, or moretimes. The collected extended capture probes from one or more releasesteps can be detected and used to correlate the first, second, third, orfourth biological analyte with the barcode of the capture probe to whichthe biological analyte was bound at a distinct spatial position of thesubstrate. In some embodiments, wherein selective capture probe cleavageis used, the substrate comprises two or more pluralities of captureprobes, e.g., a plurality of first capture probes, a plurality of secondcapture probes, a plurality of third capture probes. In someembodiments, the capture probes of a plurality (e.g., a plurality offirst capture probes) comprise a different cleavage domain from thecapture probes of another plurality (e.g., a plurality of second captureprobes). In some embodiments, members of a plurality of capture probes(e.g., a plurality of first capture probes) comprise a differentcleavage domain than members of another plurality of capture probes(e.g., a plurality of second capture probes). In some embodiments, thecapture probes of each plurality comprise a different cleavage domainfrom the capture probes of each other plurality. In some embodiments,the first capture probe is cleaved after step (b). In some embodiments,the first capture probe is cleaved before step (e). In some embodiments,members of the plurality of first capture probes are cleaved after step(b). In some embodiments, members of the plurality of first captureprobes are cleaved before step (e).

In some embodiments, the biological sample is contacted with two or moresubstrates. For example, after a subcellular region of a cell, aparticular type of cell, a region of interest of a biological sample, ora combination thereof, is permeabilized and a biological analyte fromthe subcellular region of the cell, particular type of cell, region ofinterest of the biological sample, or combination thereof, is capturedon a first substrate, the first substrate can be removed, and abiological analyte from a different subcellular region of a cell,particular type of cell, region of interest of a biological sample, orcombination thereof, can be captured on a second substrate. See, forexample, FIG. 34.

In some embodiments, after releasing at least one biological analytefrom a subcellular region of a cell, a particular type of cell, a regionof interest of a biological sample, or a combination thereof, the methodfurther comprises: (e) contacting the biological sample with a secondsubstrate comprising a plurality of capture probes, wherein a captureprobe comprises a spatial barcode and a capture domain; (f) releasing abiological analyte from a subcellular region of a cell, a particulartype of cell, a region of interest of a biological sample, or acombination thereof, e.g., a second subcellular region of a cell,particular type of cell, region of interest of a biological sample, orcombination thereof, wherein the biological analyte is bound by acapture probe at a distinct spatial position of the substrate; (g)detecting the bound biological analyte from step (e); and (h)correlating the biological analyte from step (e) with a barcode of thecapture probe at the distinct spatial position of the substrate, whereinthe subcellular region of the cell, particular type of cell, region ofinterest of the biological sample, or combination thereof, in (f) isdifferent than the subcellular region of the cell, particular type ofcell, region of interest of the biological sample, or combinationthereof in (b). In some embodiments, steps (e) through (g) are repeatedone (e.g., for a third subcellular region of the cell, particular typeof cell, region of interest of the biological sample, or combinationthereof), two (e.g., for a fourth subcellular region of the cell,particular type of cell, region of interest of the biological sample, orcombination thereof), three (e.g., for a fifth subcellular region of thecell, particular type of cell, region of interest of the biologicalsample, or combination thereof), or four (e.g., for a sixth subcellularregion of the cell, particular type of cell, region of interest of thebiological sample, or combination thereof) times. In some embodiments,steps (e) through (g) are repeated one, two, three, or four times. Insome embodiments, the subcellular region of one or more cells of step(b) is cytosol and the subcellular region of step (e) comprises at leastone of a nucleus, a mitochondria, and a microsome. In some embodiments,the biological sample is washed prior to step (e).

IX. Methods for Temporally Profiling a Biological Analyte in aBiological Sample

In some embodiments, the methods described herein can be performed onthe same biological sample or cells obtained from the subject at morethan one time points (e.g., before or after treatment with an agent,different stages of differentiation, different stages of diseaseprogression, different ages of the subject, or before or afterdevelopment of resistance to an agent). In some embodiments, thebiological sample is contacted with a different substrate at each timepoint. See, e.g., FIG. 35. In some embodiments, the amount of time thatpasses between two or more time points can be measured, for example, inseconds, minutes, days, weeks, or years. In some embodiments, themethods described herein are performed before and after treatment withan agent. In some embodiments, the methods described herein areperformed before and after one or more stages of differentiation. Insome embodiments, the methods described herein are performed before andafter stages of disease progression. In some embodiments, the methodsdescribed herein are performed before and after various ages of thesubject. In some embodiments, the methods described herein are performedbefore and after development of resistance to an agent. In someembodiments, the methods described herein are performed before and aftera cell migration assay as described in Section VII above.

In some embodiments, provided herein is a method for temporal profilingthe spatial location of at least one biological analyte present in abiological sample, including: (a) contacting a plurality of analytecapture agents to a biological sample comprising a cell disposed on asubstrate, wherein: an analyte capture agent of the plurality of theanalyte capture agents comprises (i) an analyte binding moiety thatbinds specifically to the biological analyte, (ii) an analyte bindingmoiety barcode; and (iii) an analyte capture sequence; and the substratecomprises a plurality of capture probes, wherein a capture probe of theplurality of capture proteins comprises (i) a spatial barcode, and (ii)a capture domain; (b) releasing a biological analyte from the cell,wherein the analyte capture sequence is specifically bound by thecapture domain; (c) determining (i) all or a part of the sequence of theanalyte binding moiety barcode specifically bound to the capture domain,or a complement thereof, and (ii) all or a part of the sequence of thespatial barcode or a complement thereof, and using the determinedsequences of (i) and (ii) to identify a location of the biologicalanalyte in the biological sample; and (d) repeating steps (a)-(c) aftera period of time. In some embodiments, the same biological analyte isdetected at two or more time points. In some embodiments, step (d) isrepeated one, two, three, four, or more times. In some embodiments, themethod further includes generating a temporal profile for the samebiological analyte over time. See, e.g., FIG. 37, which shows atwo-dimensional sample (e.g., a tissue sample) that is analyzed overtime (t) by examining multiple identical slides. In some embodiments,the method further includes generating a temporal profile more than onebiological analyte over time.

In some embodiments, provided herein is a method for temporal andspatial profiling of a biological analyte present in a biologicalsample, comprising: (a) contacting a biological sample with a substratecomprising a plurality of capture probes, wherein a capture probe of theplurality of capture probes comprises a spatial barcode and a capturedomain; (b) releasing a biological analyte from the biological sample,wherein the capture domain binds specifically to the released biologicalanalyte; (c) determining (i) all or a part of the sequence of thebiological analyte specifically bound to the capture domain, or acomplement thereof, and (ii) all or a part of the sequence of thespatial barcode or a complement thereof, and using the determinedsequences of (i) and (ii) to identify a location of the biologicalanalyte in the biological sample; and (d) repeating steps (b)-(c) aftera period of time.

In some embodiments, provided herein is a method for temporal andspatial profiling of a biological analyte present in a biologicalsample, comprising: (a) contacting a plurality of analyte capture agentsto a biological sample comprising a live cell, wherein the biologicalsample is disposed on a substrate, wherein: an analyte capture agent ofthe plurality of the analyte capture agents comprises (i) an analytebinding moiety that binds specifically to the biological analyte, (ii)an analyte binding moiety barcode; and (iii) an analyte capturesequence; and the substrate comprises a plurality of capture probes,wherein a capture probe of the plurality of capture proteins comprises(i) a spatial barcode, and (ii) a capture domain; (b) releasing abiological analyte from the live cell, wherein the analyte capturesequence is specifically bound by the capture domain; (c) determining(i) all or a part of the sequence of the analyte binding moiety barcodespecifically bound to the capture domain, or a complement thereof, and(ii) all or a part of the sequence of the spatial barcode or acomplement thereof, and using the determined sequences of (i) and (ii)to identify a location of the biological analyte in the biologicalsample; and (d) repeating steps (b)-(c) after a period of time.

In some embodiments, the biological sample used in the method fortemporal profiling includes a tissue, an organ, an organism, or a cellculture sample as described e.g., in Section VI herein. In someembodiments, the biological sample is in a controlled environment. Insome embodiments, the step of releasing comprises releasing underconditions that permit cells in the biological sample to live. In someembodiments, the biological sample is a subcellular fraction asdescribed herein (e.g., in Section VIII above).

The present disclosure also provides compositions and methods fortemporal and spatial profiling of at least one species of biologicalanalyte present in a biological sample that include:

(a) providing a substrate that includes a plurality of capture probes,wherein the capture probes include a spatial barcode and a capturedomain;

(b) contacting the biological sample with the solid support thatincludes the capture probes;

(c) releasing at least one member of the species of biological analytefrom the biological sample;

(d) contacting the solid support that includes the capture probes withat least one released member of the species of biological analyte suchthat the at least one released member of the species of biologicalanalyte interacts with at least one of the plurality of capture probes;

(e) detecting the at least one released member of the species ofbiological analyte;

(f) correlating the at least one released member of the species ofbiological analyte with a spatial location in the biological sample; and

(g) repeating steps (a)-(e) after a period of time.

In some embodiments, the at least one released biological analyte is anucleic acid, the capture domain is a nucleic acid, and the at least onereleased biological analyte hybridizes to the capture domain.

In some embodiments, the biological sample is in a controlledenvironment (e.g., a flow cell, a tissue culture system (e.g. a modifiedtissue culture system), or a tissue bath). In some embodiments, aremethods for temporal and spatial profiling of at least one species ofbiological analyte present in a biological sample include imaging thebiological sample (e.g., imaging can be performed prior to releasing atleast one member of the species of biological analyte from thebiological sample, after releasing at least one member of the species ofbiological analyte from the biological sample, or both). In someembodiments, imaging is used to determine a region of interest in thebiological sample. In some embodiments, imaging comprises using fiducialmarkers as disclosed herein.

In some embodiments, the step of releasing comprises applying anelectric field to the biological sample (e.g., electroporation and/orsubjecting biological sample to brief electric pulsing conditions). Insome embodiments, the electric field is applied as a short burst. Insome embodiments, the electric field is applied for a period of time ofabout 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes.In some embodiments, the electric field is applied for a period of timeof less than one minute. In some embodiments, the electric field isapplied for a period of time of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more hours. Insome embodiments, the step of releasing comprises releasing underconditions that permit cells in the biological sample to live (e.g.,conditions that prevent hypoxia or other situations that can lead tocell death).

In some embodiments, step (g) is repeated 1, 2, 3, 4, 5, 10, 15, 20, ormore times. In some embodiments, a temporal profile for the at least onemember of the species of biological analyte in the biological sample isgenerated.

In some embodiments, the same species of biological analyte is detectedat two or more time points according to any of the variety of methodsdescribed herein. In some embodiments, the two or more species ofbiological analytes are the same class of analyte (e.g., RNA, mRNA, DNA,proteins, antibodies, peptides, lipids, metabolites, or small molecules.In some embodiments, the two or more species of biological analytes aredifferent classes of analytes. In some embodiments, two or more speciesof two or more classes of biological analytes are analyzed at multipletime points to generate a temporal and spatial profile of the analytesin the biological sample.

In some embodiments, prior to the step of repeating, the biologicalsample is subjected to one or more perturbants. In some embodimentsprovided herein is a method for determining the effect of a perturbanton a biological sample, comprising: (a) contacting the biological samplewith the perturbant; and (b) performing a method for temporal profilingas described herein. In some embodiments, the perturbant comprises asmall molecule, an antibody, a nucleic acid, a peptide, or a combinationthereof.

A perturbant can be any physical molecule such as, without limitation, asmall molecule, an antibody, a nucleic acid, a peptide, or anycombination thereof. In some embodiments, a perturbant includes a DNAmodification system (e.g., a CRISPR system, e.g., a system that employsa single guide RNA (sgRNA) and an enzymatic molecule or complex (e.g.Cas9 or a derivative thereof) to modify a nucleic acid (e.g.,chromosomal DNA) of cells in the biological sample). In someembodiments, a perturbant is a perturbing condition that is not aphysical molecule. For example, a perturbant can be, without limitation,light, heat, pressure, an electric field, or any combination thereof. Insome embodiments, the biological sample is subjected to same perturbantat multiple time points (e.g., between repeating steps (a)-(f) abovesuch that the spatial and temporal profiles of at least one analyte inthe biological sample are determined after each time the biologicalsample is subjected to the perturbant). In some embodiments, thebiological sample is subjected to a different perturbant at multipletime points (e.g., between repeating steps (a)-(f) above such that thespatial and temporal profiles of at least one analyte in the biologicalsample are determined after each the biological sample is subjected tomultiple perturbants at different times). In some embodiments, both aphysical molecule and a perturbing condition are used as perturbants. Insome embodiments, a physical molecule and a perturbing condition areused simultaneously as perturbants. In some embodiments, a physicalmolecule and a perturbing condition are used sequentially asperturbants. In some embodiments, a perturbant can be used in accordancewith compositions and methods described herein to determine the effectof an agent (e.g., the perturbant can be an agent such as, withoutlimitation, a small molecule drug or biologic agent) on a biologicalsample. For example the biological sample can be a cancer sample and theagent acting as a perturbant can be a small molecule drug or biologicagent, which is tested for its effect on the cancer sample.

X. Spatially-Tagged Analyte Capture Agents for Spatial Detection

Provided herein are methods for performing immunohistochemistry and/orimmunofluorescence on a capture probe array as described herein. Furtherprovided are methods of spatially-tagging analyte capture agents with acapture probe spatial barcode. Further provided are methods for blockingthe analyte capture sequence of an analyte capture agent. Furtherprovided are methods for labeling multiple analytes using the samebiological sample. In some embodiments, the labels for the multipleanalytes are applied to the biological sample at the same time, and insome embodiments, the labels for the multiple analytes are applied tothe biological sample at different times (e.g., sequentially).

(a) Spatial Array Compatible Immunohistochemistry(IHC)/Immunofluorescence (IF)

In some embodiments, IHC and/or IF protocols can be performed on any ofthe spatial arrays provided herein. In some embodiments, a substrate canbe homogenously covered by a plurality of capture probes. In someembodiments, IHC and/or IF protocols can be performed on a biologicalsample on a spatial array, wherein after the IHC and/or IF protocol iscompleted, the biological sample is treated to release analytes forcapture by the spatial array. In some embodiments, an antibody of theIHC/IF protocol detects an extracellular protein. In some embodiments,the antibody of the IHC/IF protocol detects a cell surface protein. Insome embodiments, the antibody of the IHC/IF protocol detects anintracellular protein (e.g., GFAP, NeuN). In some embodiments, theantibody of the IHC/IF protocol detects a nuclear protein. In someembodiments, the biological sample is fixed. The biological sample canbe fixed by methods described herein, or according to methods known inthe art. In some embodiments, the biological sample can be blocked,permeabilized, and stained by methods described herein, or according tomethods known in the art. In some embodiments, IHC and/or IF protocolscan be performed using a detectable label. A detectable label caninclude radioisotopes, fluorophores, chemiluminescent compounds,bioluminescent compounds, and dyes, including any of the non-limitingexamples of detectable labels described herein. In some embodiments, adetectable label can be a fluorophore. In some embodiments, thebiological sample can be imaged according to methods described herein orknown in the art.

In some embodiments after the biological sample is imaged, thebiological sample can be processed and analyzed according to the spatialanalysis workflows described herein. In some embodiments, the IHC and/orIF antibodies are removed from the biological sample before performingthe spatial analysis workflows. In other embodiments, the IHC and/or IFantibodies are not removed from the biological sample before performingthe spatial analysis workflows.

(i) Immunofluorescence

Immunofluorescence or immunohistochemistry is a technique used toexamine protein expression, distribution, localization, and activationin situ. In some embodiments, immunofluorescence uses the specificity offluorescently labeled antibodies or probes that bind to specific targetanalytes (e.g., within or on a cell surface), and allows for theexamination of the distribution of the target analytess through thetissue sample. In some embodiments, immunofluorescence staining methodscan be divided into two categories, direct and indirect methods. In someembodiments, in the direct staining method, a fluorophore is conjugatedto an antibody (e.g., “the primary antibody”) which is capable ofbinding directly to the target analyte of interest (e.g., cell surfaceor intracellular target). In some embodiments, in the indirect stainingmethod, the primary antibody binding to the target analyte is visualizedby the binding of a fluorescently labeled second-step antibody, whereinthe second-step antibody is capable of binding to the primary antibody.In some embodiments, the second-step antibody is an anti-immunoglobulinantibody.

In some embodiments, immunofluorescence is performed individually foreach analyte of interest. In some embodiments, immunofluorescence cansimultaneously detect one or more target proteins in the same cell ortissue (e.g., multiplexed analyte detection). In some embodiments,multiplexing (e.g., simultaneously detecting multiple analytes) allowsfor examination of spatial arrangement of analytes of interest (e.g.,proteins, DNA, RNA) as well as analyte interaction and co-localizationthereby facilitating simultaneous analysis of multiple tissue markers.

(b) Spatially-Tagged Capture Agents and Methods for Using the Same

An “analyte capture agent” refers to a molecule that interacts with atarget analyte and with a capture probe to identify the analyte. In someembodiments, an analyte capture agent includes a label (e.g.,fluorescent label). In some embodiments, the analyte capture agent caninclude an analyte binding moiety and a capture agent barcode domain. Ananalyte binding moiety is a molecule capable of binding to a specificanalyte. In some embodiments, the analyte binding moiety includes anantibody or antibody fragment. In some embodiments, the analyte bindingmoiety includes a polypeptide and/or an aptamer. In some embodiments,the analyte binding moiety includes a DNA aptamer. In some embodiments,the analyte binding moiety includes a RNA aptamer. In some embodiments,the analyte binding moiety includes an aptamer of mixed natural orunnatural occurring nucleotides (e.g., LNA, PNA). In some embodiments,the analyte is a protein (e.g., a protein on a surface of a cell or anintracellular protein).

A capture agent barcode domain can include an analyte capture sequencewhich can hybridize to at least a portion or an entirety of a capturedomain of a capture probe. In some embodiments, the analyte capturesequence includes a poly (A) tail. In some embodiments, the analytecapture sequence includes a sequence capable of binding a poly (T)domain. In some embodiments, the analyte capture sequence can have a GCcontent between 1-100%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, etc.).In some embodiments, the analyte capture sequence has a GC content of atleast 30%. In some embodiments, one or more pluralities of analytecapture agents can be provided to a biological sample, wherein oneplurality of analyte capture agent differs from another plurality ofanalyte capture agent by the analyte capture sequence. For example,analyte capture sequence A can be correlated with analyte binding moietyA, and analyte capture sequence B can be correlated with analyte bindingmoiety B. In some embodiments, the two pluralities of analyte captureagents can have the same analyte binding moiety barcode sequence.

In some embodiments, the capture domain includes a poly (T) tail. Insome embodiments, the capture domain includes a sequence capable ofbinding a poly (A) domain. In some embodiments, the capture domain canhave a GC content between 1-100%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,80%, etc. In some embodiments, the capture domain has a GC content of atleast 30%.

In some embodiments, the capture agent barcode domain includes ananalyte binding moiety barcode. The analyte binding moiety barcoderefers to a barcode that is associated with or otherwise identifies theanalyte binding moiety. In some embodiments, the analyte binding moietybarcode is correlated with the type of analyte binding moiety, such thatmore than one plurality of analyte capture agents can be provided to abiological sample at one time. For example, analyte binding moietybarcode A can be correlated with analyte binding moiety A, and analytebinding moiety barcode B is correlated with analyte binding moiety B.The two pluralities of analyte capture agents can have the same analytecapture sequence (e.g., poly(A)). In some embodiments, one analytebinding moiety barcode plurality is correlated with one analyte capturesequence plurality. In other embodiments, an analyte binding moietybarcode plurality is not necessarily correlated with an analyte capturesequence plurality.

In some embodiments, a capture agent barcode domain includes optionalsequences, such as, without limitation, a PCR handle, a sequencingpriming site, a domain for hybridizing to another nucleic acid molecule,and combinations thereof. In some embodiments, the PCR handle isidentical on all capture analyte barcode domains. In some embodiments,the PCR handle is included for PCR amplification. In some embodiments,an analyte capture agent includes one or more optional sequences and oneor more barcode sequences (e.g., one or more analyte binding moietybarcodes and/or one or more UMIs). In some embodiments, the captureprobe capture domain and/or the analyte capture agent include a cleavagedomain. In some embodiments, a capture agent barcode domain can bedissociated from the analyte binding moiety by cleaving the analytebinding moiety from the capture agent barcode domain via a cleavagedomain in the capture agent barcode domain. Other embodiments of ananalyte capture agent useful in spatial protein detection are describedherein.

Provided herein are methods for spatially profiling a biologicalanalyte, e.g., any of the analytes as described herein, in a biologicalsample that use a spatially-tagged analyte capture agent. A biologicalanalyte can be bound by an analyte capture agent at a distinct spatialposition on a substrate and detected. The bound biological analyte canthen be correlated with a barcode of the capture probe at a distinctspatial position of the substrate. In some embodiments, these methodscan include spatially profiling the biological analyte from one or moreof: an intracellular region of a cell in a biological sample, a cellsurface region of a cell in a biological sample, a particular type ofcell in a biological sample, and a region of interest of a biologicalsample.

(i) Blocking Probes

In some embodiments, an analyte capture sequence of a capture agentbarcode domain is blocked prior to adding the analyte capture agent to abiological sample. In some embodiments, an analyte capture sequence of acapture agent barcode domain is blocked prior to adding the analytecapture agent to a capture probe array. In some embodiments, blockingprobes are added to blocking buffer or other solutions applied in an IHCand/or IF protocol. In some embodiments, a blocking probe is used toblock or modify the free 3′ end of the capture agent barcode domain. Insome embodiments, a blocking probe is used to block or modify the free3′ end of the analyte capture sequence of the capture agent barcodedomain. In some embodiments, a blocking probe can be hybridized to theanalyte capture sequence of a capture agent barcode domain to mask thefree 3′ end of the capture agent barcode domain. In some embodiments, ablocking probe can be a hairpin probe or partially double strandedprobe. In some embodiments, the free 3′ end of the analyte capturesequence of the capture agent barcode domain can be blocked by chemicalmodification, e.g., addition of an azidomethyl group as a chemicallyreversible capping moiety such that the capture probes do not include afree 3′ end. Blocking or modifying the capture agent barcode domains,particularly at the free 3′ end of the capture agent barcode domain,prior to contacting the analyte capture agents with the substrate,prevents binding of the analyte capture sequence to capture probecapture domain (e.g., prevents the binding of an analyte capturesequence poly(A) tail to a poly(T) capture domain).

In some embodiments, a blocking probe is used to block or modify thefree 3′ end of a capture probe. In some embodiments, a blocking probe isused to block or modify the free 3′ end of a capture probe capturedomain. In some embodiments, the analyte capture sequence is blockedprior to adding the analyte capture agent to a capture probe array. Insome embodiments, blocking probes are added to blocking buffer or othersolutions applied in an IHC and/or IF protocol. In some embodiments, ablocking probe can be hybridized to the capture domain to mask the free3′ end of the capture domain. In some embodiments, a blocking probe canbe a hairpin probe or partially double stranded probe. In someembodiments, the free 3′ end of the capture domain can be blocked bychemical modification, e.g., addition of an azidomethyl group as achemically reversible capping moiety such that the capture probes do notinclude a free 3′ end. Blocking or modifying the capture domains,particularly at the free 3′ end of the capture domain, prior tocontacting the analyte capture agents with the capture probe array,prevents binding of the analyte capture sequence to capture probecapture domain (e.g., prevents the binding of an analyte capturesequence poly(A) tail to a poly(T) capture domain).

In some embodiments, the blocking probes can be reversibly removed. Forexample, blocking probes can be applied to block the free 3′ end ofeither or both the capture agent barcode domain and/or the captureprobes. Blocking interaction between the analyte capture agent and thecapture probe array can reduce non-specific background staining in IHCand/or IF applications. After the analyte binding agents are bound tothe target analyte, the blocking probes can be removed from the 3′ endof the capture agent barcode domain and/or the capture probe, and theanalyte-bound analyte binding agents can migrate to and become bound bythe capture probe array. In some embodiments, the removal includesdenaturing the blocking probe from the analyte binding moiety barcodeand/or capture probe. In some embodiments, the removal includes removinga chemically reversible capping moiety. In some embodiments, the removalincludes digesting the blocking probe with an RNAse (e.g., RNAse H).

In some embodiments, the blocking probes are oligo (dT) blocking probes.In some embodiments, the oligo (dT) blocking probes can have a length of15-30 nucleotides. In some embodiments, the oligo (dT) blocking probescan have a length of 10-50 nucleotides, e.g., 10-50, 10-45, 10-40,10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30,15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45,25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40,40-50, 40-45, or 45-50 nucleotides. In some embodiments, the analytecapture agents can be blocked at different temperatures (e.g., 4° C. and37° C.). In some embodiments, the analyte capture agents can be blockedfrom binding to the capture probes more effectively at lowertemperatures when using shorter blocking probes.

(ii) Spatially-Tagged Capture Agents

A “spatially-tagged analyte capture agent” can be a molecule thatinteracts with an analyte (e.g., an analyte in a sample) and with acapture probe to identify the spatial location of the analyte. In someembodiments, a spatially-tagged analyte capture agent can be an analytecapture agent with an extended capture agent barcode domain thatincludes a sequence complementary to a spatial barcode of a captureprobe. In some embodiments, an analyte capture agent is introduced to ananalyte and a capture probe at the same time. In some embodiments, ananalyte capture agent is introduced to an analyte and a capture probe atdifferent times. In some embodiments, the spatially-tagged analytecapture agent is denatured from the capture probe before the biologicalsample is introduced. In some embodiments, the spatially-tagged analytecapture agent binds to a biological analyte within a biological samplebefore the spatially-tagged analyte capture agent is denatured from thecapture probe. In some embodiments, the capture probe is cleaved fromthe substrate while attached to the spatially-tagged analyte captureagent. In some embodiments, once the capture domain of the capture probeis bound to the analyte binding moiety barcode, the analyte capturesequence is extended towards the 3′ tail to include a sequence that iscomplementary to the sequence of the capture probe spatial barcode(e.g., producing a spatially-tagged analyte capture agent).

For example, an analyte capture agent can be introduced to a biologicalsample, wherein the analyte binding moiety binds to a target analyte,and then the biological sample can be treated to release theanalyte-bound analyte capture agent from the sample. The analyte-boundanalyte capture agent can then migrate and bind to a capture probecapture domain, and the analyte-bound capture agent barcode domain canbe extended to generate a spatial barcode complement at the end of thecapture agent barcode domain. The analyte-bound spatially-tagged analytecapture agent can be denatured from the capture probe, and analyzedusing methods described herein.

In another example, an analyte capture agent can be hybridized to acapture probe capture domain on a capture probe array, wherein thecapture agent barcode domain is extended to include a sequencecomplementary to the spatial barcode of the capture probe. A biologicalsample can be contacted with the analyte capture agent modified captureprobe array. Analytes from the biological sample can be released fromthe sample, migrated to the analyte capture agent modified capture probearray, and captured by an analyte binding moiety. The capture agentbarcode domain of the analyte-bound analyte capture agents can bedenatured from the capture probe, and the biological sample can bedissociated and spatially processed according to methods describedherein.

In some embodiments, a spatially-tagged analyte capture agent can attachto a surface of a cell through a combination of lipophilic and covalentattachment. For example, a spatially-tagged analyte capture agent caninclude an oligonucleotide attached to a lipid to target theoligonucleotide to a cell membrane, and an amine group that can becovalently linked to a cell surface protein(s) via any number ofchemistries described herein. In these embodiments, the lipid canincrease the surface concentration of the oligonucleotide and canpromote the covalent reaction.

In some embodiments, a splint oligonucleotide can be used to join thecapture agent barcode domain to a capture probe. A “splintoligonucleotide” is an oligonucleotide that, when hybridized to otherpolynucleotides, acts as a “splint” to position the polynucleotides nextto one another so that they can be ligated together. In someembodiments, the splint oligonucleotide is DNA or RNA. The splintoligonucleotide can include a nucleotide sequence that is partiallycomplimentary to nucleotide sequences from two or more differentoligonucleotides. In some embodiments, a splint oligonucleotide includesa sequence useful for ligating the capture agent barcode domain onto thecapture probe. For example, a capture agent barcode domain of an analytecapture agent includes a cleavage domain and an analyte binding moietybarcode, but does not include an analyte capture sequence. A splintoligonucleotide, which corresponds to a portion of the analyte bindingmoiety barcode and the capture probe capture domain, can hybridize toboth the analyte binding moiety barcode and the capture domain, allowingthe analyte binding moiety barcode to ligate to the capture probecapture domain.

In some embodiments, an analyte-bound, spatially-tagged analyte captureagent is compartmentalized into individual partitions (e.g.,spatially-tagged analytes dissociated into single cells), and barcodes(e.g., the analyte binding moiety barcode and/or the spatial barcode ofthe spatially-tagged analyte capture agent) can be sequenced, analyzed,and correlated with the target analyte. In some embodiments, thespatially-tagged analyte capture agent can be separated into two or moreparts, e.g., the spatially-tagged capture agent barcode domain (e.g.,the extended oligonucleotide conjugated to the analyte binding moiety)can be separated from the analyte-bound analyte binding moiety bydisrupting a linker that joins the two entities. In some embodiments,the spatially-tagged capture agent barcode domain is analyzed in thesame compartment as the analyte-bound analyte binding moiety. In someembodiments, the spatially-tagged capture agent barcode domain isseparated from the analyte-bound, analyte binding moiety beforecompartmentalization. In some embodiments, each partition maintainsseparation of its own contents from the contents of other partitions.For example, the partition can be a droplet in an emulsion or anon-emulsion based partitioning such as encapsulation. In someembodiments, a partition can include a biological analyte bound to ananalyte capture agent (e.g., a spatially-tagged analyte capture agent).In some embodiments, the 3′ tail of the analyte binding moiety barcode(e.g., antibody barcode) can be captured and barcoded in an emulsion. Insome embodiments, a tissue sample or tissue section is disassociatedinto single cells, reverse transcription (RT) reagents, and oil arecombined on a microfluidic chip to form emulsion partition reactionvesicles. In some embodiments, partitions are formed in parallel withinthe microfluidic channels of the chip, allowing the user to process100's to 10,000's of the single cells in a short period of time. In someembodiments, within each partition reaction vesicle, a single cell islysed and reverse transcription of polyadenylated mRNA occurs. As aresult, cDNAs from a single cell from individual partitions will retainthe same barcode, allowing sequencing reads from the cDNAs to be mappedback to their original single cell of origin in the tissue section ortissue sample.

In some embodiments, a method of associating or identifying a targetbiological analyte with a location within a biological sample caninclude providing a substrate with a plurality of capture probes (e.g.,a capture probe array), contacting the capture-probe array with aplurality of analyte capture agents, extending the capture agent barcodedomain of the analyte capture agent to include a sequence that iscomplementary to the sequence of the capture probe spatial barcode,thereby producing a partially-double stranded oligonucleotide includingthe capture domain, spatial barcode, and other optional domains of thecapture probe, as well as the analyte capture sequence, analyte bindingmoiety barcode and optional domains of the capture agent barcode domain,contacting the one or more spatially-tagged analyte capture agents withthe biological sample, and denaturing the spatially-tagged analytecapture agent from the capture probe. In some embodiments, the methodfurther includes dissociating the biological sample into plurality ofcells of the biological sample, and using the spatially-tagged analytecapture agent to identify the location of the target biological analytewithin the biological sample. See, for example, FIG. 46 and FIG. 47.

(iii) Releasing an Analyte Capture Agent from an Array

In some embodiments, capture probes can be initially associated with(e.g., bound to covalently or non-covalently) an array and then releasedfrom the array. In some embodiments, the capture probe can be releasedfrom an array through activation of the capture probe cleavage domain,as described herein. In some embodiments, capture probes can be coupledwith analyte capture agents via the capture domain of the capture probe,and the capture probes can denature from the spatially-tagged analytecapture agents allowing the capture probes to continue to be associatedwith the array.

For example, a cell surface protein of a cell can be associated with ananalyte capture agent, and the cell can be subjected to spatial analysis(e.g., any of the variety of spatial analysis methods described herein).In some embodiments, the analyte capture agent bound to the cell surfaceprotein can be bound to a capture probe (e.g., a capture probe on anarray) via a capture domain that interacts with an analyte capturesequence present on the capture agent barcode domain of the analytecapture agent. The capture agent barcode domain (including the analytebinding moiety barcode) can be extended with a polymerase using a 3′ endof the capture domain as a priming site, thus generating an extendedcapture agent barcode domain that includes the complementary sequencefor all or part of the capture probe (including a spatial barcode) aswell as the analyte binding moiety barcode (e.g., producing aspatially-tagged analyte capture agent). In some embodiments, thespatial array with the spatially-tagged analyte capture agents can becontacted with a sample, where the spatially-tagged analyte captureagent associated with the spatial array captures the target analyte. Thespatially-tagged analyte capture agent can then be denatured from thecapture probe of the spatial array, allowing the spatial array to bereused. The sample can be dissociated into non-aggregated cells (e.g.single cells) and analyzed by the single cell/droplet methods describedherein. The extended capture probe can be sequenced to obtain a nucleicacid sequence, in which the spatial barcode of the capture probe isassociated with the analyte binding moiety barcode of the analytecapture agent.

In some embodiments, the capture probe can be released from the arraywithout denaturing from the spatially-tagged analyte capture agent. Insome embodiments, release of the capture probes occurs upon applicationof a stimulus that initiates dissociation of the capture probes from thearray. The stimulus can be a photo-stimulus, e.g., through cleavage of aphoto-labile linkage that releases the capture probes, an elevation ofthe array's temperature (e.g., resulting in linkage cleavage or anotherrelease mechanism of the capture probes from the array). In someembodiments, a chemical stimulus can be used to cleave a linkage, orotherwise release the capture probes from the array. In someembodiments, such compositions include the polyacrylamide matrices, andcan be degraded for release of the attached capture probes throughexposure to a reducing agent, such as DTT. Additional, non-limitingstimulus examples can include, for example, a photo-stimulus, amechanical stimulus, a radiation stimulus, a biological stimulus (e.g.,enzyme), or any combination thereof, as discussed elsewhere herein.

(c) Multiplexed Analyte Analysis

In some embodiments, the arrays and methods described herein supportmultiplexed spatial analysis. In some embodiments, spatial analysis of abiological analyte can be performed individually for each analyte ofinterest. In some embodiments, multiple biological analytes can bedetected and spatially analyzed simultaneously within the samebiological sample. In some embodiments, multiplexing (e.g.,simultaneously detecting multiple markers) allows for examination ofspatial arrangement of analytes of interest (e.g., proteins, DNA, RNA)as well as analyte interaction and co-localization thereby facilitatingsimultaneous analysis of multiple tissue markers. In some embodiments,multiple biological analytes can be detected and spatially analyzed atdifferent times (e.g., detection and analysis of one category ofanalyte, followed by detection and analysis of another category ofanalyte, or detection and analysis of one analyte followed by detectionand analysis of another analyte). For example, detection, examination,and analysis of one or more proteins within a biological sample canoccur prior to the detection, examination, and analysis of one or moremRNA transcripts.

In some embodiments, as described above, the analyte binding moietybarcode can be correlated with, associated with, or correspond to aparticular analyte binding moiety, and thus a particular analyte. Forexample, analyte binding moiety A can correspond to analyte bindingmoiety barcode A, and analyte binding moiety B can correspond to analytebinding moiety barcode B. In this way, a number (n), of analytes can beexamined by introducing (n) different analyte binding moiety/analytebinding moiety barcode pluralities to the sample. In some embodiments,analyte capture agents can be used in multiplexed methods to capture 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ormore analytes. In some embodiments, all analyte capture agentpluralities possess the same analyte capture sequence, and thus can becaptured by a capture probe array, wherein the capture probes possessthe same capture domain.

In some embodiments, as described above, the analyte capture sequencecan be correlated with, associated with, or correspond to a particularanalyte binding moiety, and thus a particular analyte. For example,analyte capture sequence A can correspond to analyte binding moietybarcode A, and analyte capture sequence B can correspond to analytebinding moiety barcode B. In this way, a number (n), analytes can beexamined by introducing (n) different analyte binding moiety/analytecapture sequence pluralities to the sample. In some embodiments, theanalyte binding moiety barcode and the analyte capture sequence can becorrelated with, associated with, or correspond to a particular analytebinding moiety.

In some embodiments, a particular type or category of biologicalanalytes is correlated with, associated with, or corresponds to aparticular capture domain on a capture probe array. For example, acapture probe array can be generated wherein a plurality of the captureprobes on the array possess a homopolymer (e.g., poly(T)) capture domainwhich corresponds to one type of biological analyte (e.g. mRNA).Additionally, the same capture probe array can include a plurality ofcapture probes that possess a N-mer priming sequence capture domain foranother type of biological analyte (e.g., genomic DNA), and the samecapture probe array can include a plurality of capture probes thatpossess a capture domain that can specifically bind the sequence of ananalyte capture sequence (e.g., corresponding to a third type ofanalyte, e.g., proteins).

In some embodiments of any of the spatial analysis methods describedherein, multiple (e.g., two or more, three or more) classes (e.g. types,categories) of biological analytes can be concurrently measured. Forexample, a feature can include capture probes that can participate in anassay of at least three different types of analytes via three differentcapture domains. For example, a feature can be coupled to a barcodedcapture probe that includes a capture domain that includes a poly(T)priming sequence for mRNA analysis; a barcoded capture probe thatincludes a capture domain that includes a random N-mer priming sequencefor gDNA analysis; and a barcoded capture probe that includes a capturedomain that can specifically bind an analyte capture agent (e.g., anantibody with a spatial barcode), via its analyte capture sequence.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of multiple biological analytes that can beconcurrently measured include, without limitation: (a) mRNA, a lineagetracing construct, and cell surface and/or intracellular proteins and/ormetabolites; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq,and/or MNase-seq), and cell surface and/or intracellular proteins and/ormetabolites; (c) mRNA, genomic DNA, and a perturbation reagent (e.g., aCRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein); (d) mRNA, accessible chromatin,and a perturbation reagent; (e) mRNA, an analyte capture agent (e.g.,any of the MHC multimers described herein), and a perturbation reagent;(f) mRNA, cell surface and/or intracellular proteins and/or metabolites,and a perturbation agent; (g) mRNA, a V(D)J sequence of an immune cellreceptor (e.g., T-cell receptor), and a perturbation reagent; (h) mRNA,an analyte capture agent, and a V(D)J sequence of an immune cellreceptor; (i) cell surface and/or intracellular proteins and/ormetabolites, a an analyte capture agent (e.g., the MHC multimersdescribed herein), and a V(D)J sequence of an immune cell receptor; (j)methylation status, mRNA, and cell surface and/or intracellular proteinsand/or metabolites; (k) mRNA, chromatin (e.g., spatial organization ofchromatin in a cell), and a perturbation reagent; (l) a V(D)J sequenceof an immune cell receptor, chromatin (e.g., spatial organization ofchromatin in a cell); and a perturbation reagent; and (m) mRNA, a V(D)Jsequence of an immune cell receptor, and chromatin (e.g., spatialorganization of chromatin in a cell), or any combination thereof.

In some embodiments, nucleic acids from a spatial array are amplifiedusing any of the methods disclosed herein. The resulting amplicons canbe subsequently assayed as set forth below. In some embodiments,amplicon barcodes are analyzed using multiplex spatial imaging. In someembodiments, amplicon barcodes can be analyzed by RNA sequential probingof targets (RNA SPOTs), sequential fluorescent in situ hybridization(SeqFISH), single-molecule fluorescent in situ hybridization (smFISH),multiplexed error-robust fluorescence in situ hybridization (MERFISH),in situ sequencing (ISS), targeted in situ sequencing, fluorescent insitu sequencing (FISSEQ), or spatially-resolved transcript ampliconreadout mapping (STARmap). In some embodiments, amplicon barcodes areanalyzed by sequential hybridization and detection with a plurality oflabelled probes. A variety of light-based sequencing technologies areknown in the art (Landegren et al., Genome Res. 8:769-76 (1998); Kwok,Pharmocogenomics 1:95-100 (2000); and Shi, Clin. Chem. 47:164-172(2001), each herein incorporated by reference in its entirety). In someembodiments, the methods provided herein involves capturing a targetnucleic acid using barcoded capture probes, and optionally generatingcDNA molecules that include the barcodes from the capture probes. Insome embodiments, labeled probes (e.g., fluorescently labeled probes, orfluorophore-conjugated detection oligonucleotides) are used to sequencethe barcodes from the capture probes, thereby visualizing the locationof the target nucleic acid. In some embodiments, a labeled probe islabeled with a detectable moiety. In some embodiments, a labeled probeincludes one detectable moiety. In some embodiments, a labeled probeincludes two or more detectable moieties. In some embodiments, adetectable moiety is or includes a fluorophore. In some embodiments, thebarcodes from the capture probes are sequenced by introducingfluorophores in a base-specific manner. In some embodiments, an image istaken after each addition of a fluorophore. In some embodiments,provided methods include a step of removing labeled probes after eachimaging step. In some embodiments, a step of removing includes degradingthe labeled probes. In some embodiments, a step of removing does notsignificantly degrade a labeled probe. In some embodiments, labeledprobes (e.g., fluorescently labeled probes, or fluorophore-conjugateddetection oligonucleotides) are used to visualize the location of atarget RNA directly, without the generation of cDNA molecules.

Exemplary probes labeled with fluorophores include but are not limitedto probes for fluorescence in situ hybridization (FISH). Widely knownand practiced by persons having ordinary skill in the art, FISH is usedto, among other things, detect and localize the presence or absence ofspecific DNA sequences or RNA targets. Methods for designing andpreparing labeled probes are widely known in the art, including but notlimited to those described in US 2012/0142014.

Library Preparation for Protein Detection

(c) Biological Samples and Analytes

It has been identified that any of the methods described herein can beused for various types of biological samples. Further, profiles ofbiological samples can be compared to profiles of other cells, e.g.,“normal,” or “healthy,” biological samples. Thus, in some embodiments ofany the methods described herein, the methods can provide for diagnosisof a disease. In some embodiments of any the methods described herein,the methods can be used in drug screening.

In some embodiments, the biological sample is an organism. In someembodiments, the biological sample is an organoid. In some embodiments,the biological sample is one or more cells from a cell culture (e.g.,suspended cells or adherent cells). In some instances, the biologicalsample is a plurality of live cells (e.g., from a cell culture (e.g.,suspended cells or adherent cells)). In some embodiments, the biologicalsample is a tissue section.

In some embodiments, the thickness of the tissue section is at least 10micrometers. For example, the thickness of the tissue section can be atleast 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 13, 14, 15, 20, 30, 40, or 50 micrometers. Thicker sections canalso be used if desired or convenient, e.g., at least 70, 80, 90, or 100micrometers or more. Typically, the thickness of a tissue section isbetween 1-100 micrometers, 1-50 micrometers, 1-30 micrometers, 1-25micrometers, 1-20 micrometers, 1-15 micrometers, 1-10 micrometers, 2-8micrometers, 3-7 micrometers, or 4-6 micrometers, but as mentionedabove, sections with thicknesses larger or smaller than these ranges canalso be analysed.

In some embodiments, the biological sample is derived from a homogenouscellular population. In some embodiments, the biological sample isderived from a heterogeneous cellular population. In some embodiments,the heterogeneous cellular population is a culture of cells, a wholeorganism, or an organoid.

In some embodiments, the biological sample is a cell culture. A cellculture as described herein can comprise a plurality of cells. In someembodiments, the plurality of cells is from a cell culture of suspendedcells or a cell culture of adherent cells as described herein. Forexample, the adherent cells can be from cell lines comprising at leastone of BT549, HS 578T, MCF7, MDA-MB-231, MDAMB-468, T-47D, SF268, SF295,SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29,KM12, SW620, 786-O, A498, ACHN, CAKI, RXF 393, SN12C, TK-10, UO-31,A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, LOXIMVI, M14, MALME-3M, MDA-MB-435, SK-, EL-2, SK-MEL-28, SK-MEL-5,UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3,NCI-ADR-RES, DU145, PC-3, DU145, H295R, HeLa, KBM-7, LNCaP, MCF-7,MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D, THP-1, U87, vero, MC3T3, GH3,PC12, dog MDCK kidney epithelial, Xenopus A6 kidney epithelial,zebrafish AB9, or Sf9 insect epithelial cell lines. In some embodiments,the biological sample is a plurality of cells from a disassociatedtissue or a tissue section. In some embodiments, the biological sampleis a plant. In some embodiments, the plant is a monocotyledonous plant(i.e., a monocot). For example, a monocotyledonous plant includes but isnot limited to corn, rice, sugarcane, switchgrass, meadow grass species,Chinese sorghum, barley, wheat and oat, and durum wheat. In someembodiments, the plant is a dicotyledonous plant (i.e., a dicot). Forexample, a dicotyledonous plant includes but is not limited to soybean,cotton, rape, flax, sunflower, legume, clover, peanut, and tomato.

In some embodiments, the biological samples described herein includeanalytes of interest. In some embodiments, one or more biologicalanalytes are detected and analyzed in a biological sample. In someembodiments, the analyte is any analyte as described herein, includingbut not limited to a nucleic acid, a lipid, a carbohydrate, or aprotein. In some embodiments, the analyte is an organelle as describedherein.

In some embodiments, the analyte can be a cell surface marker. In someembodiments, a plurality of analyte capture agents can be disposed onthe surface of a substrate as described herein. In some embodiments, theplurality of analyte capture agents to a biological sample can be anantibody. In some embodiments, the plurality of analyte capture agentsto a biological sample can bind to a cell surface marker. Non-limitingexamples of a cell surface marker include CD45, CD3, CD4, CD8, CD56,CD19, CD20, CD11 c, CD14, CD33, CD66b, CD34, CD41, CD61, CD235a, CD146,and epithelial cellular adhesion molecule (EpCAM). An analyte captureagent disposed on the surface of a substrate can be used to bind a cellthat has the corresponding cell surface maker.

In some embodiments, the biological sample is contacted with a substratewhen the biological sample is in a bulk solution. In some embodiments,the bulk solution permeabilizes one or more membranes of a biologicalsample, allowing access to a biological analyte. A bulk solution can beany bulk solution as described herein.

Tissues and cells can be obtained from any source. For example, tissuesand cells can be obtained from single-cell or multicellular organisms(e.g., a mammal). Tissues and cells obtained from a mammal, e.g., ahuman, often have varied analyte levels (e.g., gene and/or proteinexpression) which can result in differences in cell morphology and/orfunction. The position of a cell or a subset of cells (e.g., neighboringcells and/or non-neighboring cells) within a tissue can affect, e.g.,the cell's fate, behavior, morphology, and signaling and cross-talk withother cells in the tissue. Information regarding the differences inanalyte levels (gene and/or protein expression) within different cellsin a tissue of a mammal can also help researchers identify and elucidateanalyte levels in the single-cell or multicellular organisms (e.g., amammal) based on the detected differences in analyte levels withindifferent cells in the tissue. Differences in analyte levels withindifferent cells in a tissue of a mammal can also provide information onhow tissues (e.g., healthy and diseased tissues) function and/ordevelop. Differences in analyte levels within different cells in atissue of a mammal can also provide information of different mechanismsof disease pathogenesis in a tissue and mechanism of action of atherapeutic treatment within a tissue. Differences in analyte levelswithin different cells in a tissue of a mammal can also provideinformation on drug resistance mechanisms and the development of thesame in a tissue of a mammal. Differences in the presence or absence ofanalytes within different cells in a tissue of a multicellular organism(e.g., a mammal) can provide information on drug resistance mechanismsand the development of the same in a tissue of a multicellular organism.

(d) Substrate(s) for Protein Detection

One or more substrates can be used in the methods as described herein.In some embodiments, a biological sample can be disposed upon asubstrate comprising a plurality of capture probes as described herein.In some embodiments, wherein one substrate is used, a biological sampleis disposed upon a substrate comprising a plurality of capture probes asdescribed herein (i.e., onto the same substrate).

In some embodiments, the substrate is contacted with the biologicalsample. In some embodiments, the biological sample is lysed orpermeabilized, allowing capture probes to associate with (e.g.,hybridize to) a target analyte. In some embodiments, after associationof the capture probe with an analyte, further steps, including cDNAsynthesis and purification, and sequencing are performed using methodsdescribed herein.

In some embodiments, a plurality of cells (e.g., a cell culturecomprising a plurality of cells) is distributed onto the substrate. Insome embodiments, wherein the substrate comprises a plurality offeatures, a cell of the plurality of cells is distributed onto thesubstrate such that the cell occupies a distinct feature on thesubstrate. In some embodiments, wherein the substrate comprises aplurality of features, members of the plurality of cells are distributedover the substrate until about all of the features on the substrate arebound to a cell. In some embodiments, wherein the substrate comprises aplurality of features, the features are smaller than the minimum celldiameter. In some embodiments, wherein the substrate comprises aplurality of features, cells not bound to a feature on the substrate arewashed off. In some embodiments, cells not bound to a feature on thesubstrate are washed off prior to releasing the biological analyte. Insome embodiments, members of the plurality of cells are distributed overthe substrate prior to the binding of members of the plurality of cellsto members of the plurality of capture antibodies.

In some embodiments, before the biological sample is distributed onto asubstrate, it is permeabilized to allow access to one or more biologicalanalytes. In some embodiments, after the biological sample isdistributed onto a substrate, it is permeabilized to allow access to oneor more biological analytes. Permeabilization can be performed using anyreagents and techniques described herein. In some embodiments, thereleasing the biological analyte comprises permeabilizing the biologicalsample, e.g., using any of the permeabilization methods as describedherein. In some embodiments, a method as described herein furthercomprises fixing the biological sample prior to permeabilization.

In some embodiments, the methods further comprise imaging the biologicalsample, e.g., imaging using any of the methods as described herein.

(e) Reagents and Buffers

In some embodiments, reagents and buffers as described herein can beused for library preparation for protein detection. For example, thereagents and buffers can include, but are not limited to, a 2× blockingbuffer, an antibody staining mix, a 1× blocking buffer, a washingbuffer, a mounting medium, a permeabilization mix, a cDNA amplificationmix, and/or a PCR reaction.

In some embodiments, a 2× blocking buffer comprises 6×SSC, 0.2% TritonX, 4% BSA, 2 U/μl RNAse inhibitor, and water. In some embodiments, the2× blocking buffer further comprises sheared salmon sperm. In someembodiments, an antibody staining mix comprises 3×SSC, 0.1% Triton X, 2%BSA, 2 U/μl RNAse inhibitor, 20 μM blocking oligos (e.g., dT25),fluorescent antibodies, and water. In some embodiments, the antibodystaining mix further comprises TotalSeq A antibody pool. In someembodiments, the antibody staining mix is kept on ice protected fromlight until use. In some embodiments, a 1× blocking buffer comprises3×SSC, 0.1% Triton X, 2% BSA, 1 U/μl RNAse inhibitor, ribonucleosidevanadyl complex, Fc block, and water. In some embodiments, a washingbuffer comprises 3×SSC, 0.1% Triton X, 2% BSA, 1 U/μl RNAse inhibitor,ribonucleoside vanadyl complex, and water. In some embodiments, amounting medium comprises 90% glycerol and 2 U/μl RNAse inhibitor. Insome embodiments, the permeabilization mix comprises tissue removalenzyme, 3×SSC, and 10% SDS. In some embodiments, a cDNA amplificationmix comprises eluted sample, amplification mix, cDNA primers, andadditive primer. In some embodiments, a PCR reaction comprises purifiedantibody-product fraction, amplification mix, TruSeq small RNA RPIxprimer, and SI-PCR primer.

(f) Tissue-optimization

Provided herein are methods for optimizing fluorescently-labelledantibody staining of a biological sample, the method comprising: (a)contacting a biological sample with a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises aspatial barcode and a capture domain; (b) fixing the biological sample;(c) rehydrating and blocking the biological sample with a blockingbuffer; (d) staining the biological sample with an antibody stainingmix, wherein the antibody staining mix comprises fluorescently labelledantibodies; (e) washing the biological sample with a washing buffer; and(f) imaging the biological sample to evaluate the quality of fluorescentantibody staining.

In some embodiments, fixing the biological sample comprises using 1%formaldehyde or 100% methanol. For example, the biological sample can beincubated in 1% formaldehyde for 10 minutes at room temperature, thenplaced in 100% methanol at −20° C. and incubated for 30 minutes orlonger. In another example, the biological sample can be fixed byincubating the biological sample in 100% methanol at −20° C. for 30minutes or longer. In some embodiments, the staining step (d) isprotected from light.

In some embodiments, the method further comprises treating thebiological sample with a permeabilization mix to permeabilize thebiological sample and release the fluorescently labelled antibodies. Insome embodiments, the biological sample is treated with thepermeabilization mix for 3, 6, 9, 12, or 18 minutes.

(g) Library Preparation Workflow for Protein Detection

In some embodiments, DNA-barcoded antibodies are used to detect proteinsin a biological sample. Provided herein are methods of preparing anantibody library from a biological sample, the method comprising: (a)contacting a biological sample with a plurality of capture probes,wherein a capture probe of the plurality of capture probes comprises aspatial barcode and a capture domain; (b) fixing and staining thebiological sample, wherein the biological sample is stained withfluorescently-labelled antibodies; (c) imaging the biological sample todetect fluorescent antibody staining; (d) permeabilizing the biologicalsample; (e) synthesizing a second strand and performing cDNAamplification; (f) selecting antibody-product for antibody sequencing;and (g) sequencing selected antibody products.

In some embodiments, the capture probes comprise an array. In someembodiments, immunohistochemistry (IHC) and/or immunofluorescence (IF)protocols can be performed for the imaging step (c) to detectfluorescent antibody staining. In some embodiments, IHC and/or IFprotocols can be performed on a biological sample on a spatial array,wherein after the IHC and/or IF protocol is completed, the biologicalsample is treated to release analytes for capture by the spatial array.In some embodiments, a fluorescently labelled antibody of the IHC/IFprotocol detects an extracellular protein. In some embodiments, thefluorescently labelled antibody of the IHC/IF protocol detects a cellsurface protein. In some embodiments, the fluorescently labelledantibody of the IHC/IF protocol detects an intracellular protein (e.g.,GFAP, NeuN). In some embodiments, the fluorescently labelled antibody ofthe IHC/IF protocol detects a nuclear protein. In some embodiments, thebiological sample is fixed. The biological sample can be fixed bymethods described herein, or according to methods known in the art. Insome embodiments, the biological sample can be blocked, permeabilized,and stained by methods described herein, or according to methods knownin the art. In some embodiments, IHC and/or IF protocols can beperformed using a detectable label. A detectable label can includeradioisotopes, fluorophores, chemiluminescent compounds, bioluminescentcompounds, and dyes, including any of the non-limiting examples ofdetectable labels described herein. In some embodiments, a detectablelabel can be a fluorophore. In some embodiments, the biological samplecan be imaged according to methods described herein or known in the art.

In some embodiments after the biological sample is imaged, thebiological sample can be processed and analyzed according to the spatialanalysis workflows described herein. In some embodiments, thefluorescently labelled antibodies are removed from the biological samplebefore performing the spatial analysis workflows. In other embodiments,the fluorescently labelled antibodies are not removed from thebiological sample before performing the spatial analysis workflows.

As used herein, immunohistochemistry or immunofluorescence is atechnique used to examine protein expression, distribution,localization, and activation in situ. In some embodiments,immunofluorescence uses the specificity of fluorescently labeledantibodies or probes that bind to specific biological analytes (e.g.,within or on a cell surface), and allows for the examination of thedistribution of the biological analytes through the tissue sample.

In some embodiments, immunofluorescence staining methods can be dividedinto two categories, direct and indirect methods. In some embodiments,in the direct staining method, a fluorophore is conjugated to anantibody (e.g., “the primary antibody”) which is capable of bindingdirectly to the biological analyte of interest (e.g., cell surface orintracellular target). In some embodiments, in the indirect stainingmethod, the primary antibody binding to the biological analyte isvisualized by the binding of a fluorescently labeled second-stepantibody, wherein the second-step antibody is capable of binding to theprimary antibody. In some embodiments, the second-step antibody is ananti-immunoglobulin antibody.

In some embodiments, immunofluorescence is performed individually foreach analyte of interest. In some embodiments, immunofluorescence cansimultaneously detect one or more target proteins in the same cell ortissue (e.g., multiplexed analyte detection). In some embodiments,multiplexing (e.g., simultaneously detecting multiple analytes) allowsfor examination of spatial arrangement of analytes of interest (e.g.,proteins, DNA, RNA) as well as analyte interaction and co-localizationthereby facilitating simultaneous analysis of multiple tissue markers.

XI. Additional Methods for Preparing Biological Samples andImmunostaining

A biological sample can be fixed in any of a variety of fixatives topreserve the biological structure of the sample prior to analysis.Application of a fixation agent to a biological sample can inducecovalent bonding interactions such as crosslinking between biomoleculesin a sample. In some embodiments, the cross-links occur betweennucleotides. In some embodiments, the cross-links occur betweennucleotides and proteins. In some embodiments, the cross-links occurbetween proteins. A sample can be fixed via immersion in ethanol,methanol, acetone, formaldehyde (e.g., 2% formaldehyde),paraformaldehyde, paraformaldehyde-Triton, glutaraldehyde, imidoesters,NHS (N-Hydroxysuccinimide) esters, or combinations thereof.

In some embodiments, a biological sample can be fixed. For example, abiological sample can be fixed by contacting the biological sample withmethanol for about 20 minutes to about 1 hour at about −30° C. to about0° C. and placing the biological sample on a substrate, where thesubstrate includes a plurality of capture probes, where a capture probeof the plurality of capture probes includes a spatial barcode and acapture domain that binds specifically to an analyte from the biologicalsample. In some embodiments, placing the biological sample on thesubstrate is performed prior to fixing the biological sample inmethanol. In some embodiments, fixing the biological sample is performedprior to placing the biological sample on the substrate.

In some embodiments, after fixing the biological sample and placing thebiological sample on the substrate the biological sample can be stained.For example, the staining can be H&E staining or immunostaining.

(a) Biological Sample Preparation

In some embodiments, a biological sample can be prepared for staining orfor spatial analysis by fresh freezing the biological sample. Forexample, fresh tissue can be isolated from a subject and/or biologicalsample and flash frozen by immersing the biological sample in isopentanechilled in liquid nitrogen. Once flash frozen, the biological sample canbe moved to a petri dish or other support and covered with OCT embeddingmedium. Once the sample and OCT are frozen, the biological sample can betransferred to a cryostat for tissue sectioning. The biological samplecan be sliced/sectioned according to the methods described in SectionI(d)(ii)(1)-(2), and the sections can be placed on a slide (e.g., aspatially-barcoded array) and warmed in order to promote sample-slideadherence. The biological sample is ready for fixing, staining, spatialanalysis, or any other workflow described herein.

In some embodiments, the biological sample is a tissue sample. In someembodiments, the biological sample is an embedded tissue sample. In someembodiments, the biological sample is an OCT embedded tissue sample. Insome embodiments, the biological sample is embedded before either fixingthe biological sample or placing the biological sample on the substrate.In some embodiments, the embedded biological sample (e.g., tissuesample) is cryosectioned.

In some embodiments, the biological sample is a tissue section. In someembodiments, the biological sample (e.g., a tissue) is perfused and thena tissue section is generated from the perfused tissue.

(b) Methanol Fixation

FIG. 54 provides a general workflow for methanol fixation of tissues.Methanol fixation can be performed in conjunction with the stainingprotocols described herein. Methanol fixation can be performed withimmunohistochemical (IHC) stains, immunofluorescence (IF) stains, or anyof the spatial analysis workflows described herein. Methanol can be usedto fix any biological sample described herein, e.g., any of thebiological samples described in Section I(d)(i). Methanol used to fixbiological samples can be 100% methanol or less than 100% methanol. Forexample, a commercial source of methanol useful in the disclosed methodscan be a 99.9% methanol solution (Millipore Sigma, HPLC grade).

Additionally, in some embodiments, fixing the biological sample (e.g.,any of the biological samples described herein) comprises contacting thebiological sample in methanol for about 1 minute to about 12 hours(e.g., about 1 minute to about 10 hours, about 1 minute to about 8hours, about 1 minute to about 6 hours, about 1 minute to about 4 hours,about 1 minute to about 2 hours, about 1 minute to about 60 minutes,about 10 minutes to about 12 hours, about 10 minutes to about 10 hours,about 10 minutes to about 8 hours, about 10 minutes to about 6 hours,about 10 minutes to about 4 hours, about 10 minutes to about 2 hours,about 10 minutes to about 1 hour, about 20 minutes to about 12 hours,about 20 minutes to about 10 hours, about 20 minutes to about 8 hours,about 20 minutes to about 6 hours, about 20 minutes to about 4 hours,about 20 minutes to about 2 hours, about 20 minutes to about 1 hour,about 20 minutes to about 40 minutes, about 40 minutes to about 12hours, about 40 minutes to about 10 hours, about 40 minutes to about 8hours, about 40 minutes to about 6 hours, about 40 minutes to about 4hours, about 40 minutes to about 2 hours, about 40 minutes to about 1hour, about 1 hour to about 12 hours, about 1 hour to about 10 hours,about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 1hour to about 2 hours, about 2 hours to about 12 hours, about 2 hours toabout 10 hours, about 2 hours to about 8 hours, about 2 hours to about 6hours, about 2 hours to about 4 hours, about 4 hours to about 12 hours,about 4 hours to about 10 hours, about 4 hour to about 8 hours, about 4hours to about 6 hours, about 6 hours to about 12 hours, about 6 hoursto about 10 hours, about 6 hours to about 8 hours, about 8 hours toabout 12 hours, about 8 hours to about 10 hours, or about 10 hours toabout 12 hours). Fixing a biological sample can be achieved bycontacting the biological sample in methanol for any range of timeperiods therein (e.g., about 1 minute to about 60 minutes, 1 minute to50 minutes, 1 minute to 40 minutes, 1 minute to 30 minutes, 1 minute to20 minutes, 10 minutes to 60 minutes, 20 minute to 60 minutes, 30minutes to 60 minutes, 40 minutes to 60 minutes, 10 minutes to 60minutes, 20 minutes to 50 minutes, 20 minutes to 40 minutes, or any subrange within these ranges). In some embodiments, fixing a biologicalsample comprises contacting (e.g., immersing) the biological sample inmethanol for about a specific time period (e.g., about 5 minutes, about10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about50 minutes, about 55 minutes, or about 60 minutes).

In some embodiments, fixing a biological sample in methanol includesprechilling the methanol. Prior to fixing a biological sample, themethanol can be chilled to between about −80° C. to about 10° C. Fixinga biological sample can be achieved by contacting the biological samplein prechilled methanol of any temperature range therein (e.g., −80° C.to about 10° C., −80° C. to about 0° C., −80° C. to about −10° C., −80°C. to about −20° C., −80° C. to about −30° C., −70° C. to about 10° C.,−60° C. to about 10° C., −50° C. to about 10° C., −40° C. to about 10°C., −30° C. to about 10° C., −70° C. to about 5° C., −60° C. to about 0°C., −50° C. to about −10° C., −40° C. to about −20° C., −40° C. to about0° C., −30° C. to about −10° C.). In some embodiments, fixationcomprises contacting (e.g., immersing) the biological sample in methanolat an approximate temperature (e.g., about −80° C., about −75° C., about−70° C., about −65° C., about −60° C., about −55° C., about −50° C.,about −45° C., about −40° C., about −35° C., about −30° C., about −25°C., about −20° C., about −15° C., about −10° C., about −5° C., about 0°C., about 5° C., about 10° C., and about 15° C.).

Methanol fixation can be combined with other tissue preservationmethods. For example, methanol fixation can be combined with freshfrozen preservation of tissues. In this case, fresh tissue can beisolated from a subject and/or biological sample and flash frozen byimmersing the biological sample in isopentane chilled in liquidnitrogen. Once flash frozen, the biological sample can be moved to apetri dish or other support and covered with OCT embedding medium. Oncethe sample and OCT are frozen, the biological sample can be transferredto a cryostat for tissue sectioning. The biological sample can besliced/sectioned according to the methods described in SectionI(d)(ii)(1)-(2), and the sections can be placed on a slide (e.g., aspatially-barcoded array) and warmed in order to promote sample-slideadherence. The sample-mounted slides can be stored (e.g., at −80° C.)until use. When ready for use, the sample-mounted slides are removedfrom storage and allowed to dry at 37° C. for 1 minute. The biologicalsample can then be fixed in methanol under any of the conditionsdescribed above. For example, the sample can be immersed in pre-chilled−20° C. methanol for 30 minutes. After methanol fixation, the biologicalsample can be subject to any of the staining or spatial analysisworkflows described herein.

(c) Aldehyde Fixation

FIG. 54 provides a general workflow for aldehyde fixation of tissues.Aldehyde fixation can be performed in conjunction with the stainingprotocols described herein. Aldehyde fixation can be performed withimmunohistochemical (IHC) stains, immunofluorescence (IF) stains, or anyof the spatial analysis workflows described herein. Aldehydes can beused to fix any biological sample described herein, e.g., any of thebiological samples described in Section I(d)(i). Non-limiting examplesof aldehydes include formaldehyde, paraformaldehyde, glutaraldehyde, andcombinations thereof. More than one fixation reagent can be used incombination in preparing a fixed biological sample. For example, in somecases biological samples such as tissue specimens are contacted with afixation reagent containing both formaldehyde and glutaraldehyde, andthus the biological samples can include fixation crosslinks resultingboth from formaldehyde induced fixation and glutaraldehyde inducedfixation.

Aldehydes are crosslinking fixatives in that they create covalentchemical bonds between proteins in tissue. Crosslinking fixatives can behelpful in preserving transient or fine cytoskeletal structure againstdegradation. A number of methods can be employed for aldehyde fixationof biological tissues. For example, the biological sample can beimmersed in an aldehyde prior to tissue sectioning (e.g., dropfixation), the biological sample can be sectioned, and the sectionimmersed in an aldehyde (e.g., on-slide fixation), or a subject (e.g.,rodent) can be perfused with an aldehyde, fixing the subject's tissuesbefore dissection (see Section VI(c)(ii)).

(i) Immersion/Drop Fixation

In some embodiments, fixing the biological sample comprises contacting(e.g., immersing) the biological sample in an aldehyde for about 1minute to about 30 minutes. Fixing a biological sample can be achievedby immersing the biological sample in an aldehyde for any range of timeperiods therein (e.g., 1 minute to 30 minutes, 1 minute to 25 minutes, 1minute to 20 minutes, 1 minute to 15 minutes, 5 minutes to 25 minutes, 5minute to 20 minutes, 5 minutes to 15 minutes, 6 minutes to 14 minutes,7 minutes to 13 minutes, 8 minutes to 12 minutes, 9 minutes to 11minutes, or any sub range within these ranges). In some embodiments,fixing a biological sample comprises contacting the biological sample inan aldehyde for about a specific time period (e.g., about 1 minute,about 2 minutes, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, or 60 minutes).

In some embodiments, fixing the biological sample comprises contacting(e.g., immersing) the biological sample in an aldehyde for about 5minute to about 20 hours. In some embodiments, fixing the biologicalsample comprises contacting (e.g., immersing) the biological sample inan aldehyde for about 1 hour to about 24 hours. In some embodiments,fixing the biological sample comprises contacting (e.g., immersing) thebiological sample in an aldehyde for about 5 hours to about 20 hours. Insome embodiments, fixing the biological sample comprises contacting(e.g., immersing) the biological sample in an aldehyde for about 10hours to about 20 hours. In some embodiments, fixing the biologicalsample comprises contacting (e.g., immersing) the biological sample inan aldehyde for about 15 hours to about 20 hours. In some embodiments,fixing the biological sample comprises contacting (e.g., immersing) thebiological sample in an aldehyde for about 1 hour, about 2 hours, about3 hours, about 4 hours, about 5 hours about 6 hours, about 7 hours,about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours,about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21hours, about 22 hours, about 23 hours, and about 24 hours. In someembodiments, fixing a biological sample in an aldehyde includesselecting the optimal fixation concentration. In some embodiments, thealdehyde is prepared in a buffer solution, wherein the aldehyde isbetween 0.1% and 10% the solution, or any range of concentration therein(e.g., 0.1% to 1%, 0.1% to 2%, 0.1% to 4%, 0.5% to 4%, 1% to 4%, 2% to4%, 2% to 5%, 1% to 5%, 2% to 10%, or any subranges therein). Forexample, aldehyde fixatives can be used at various concentrations,including 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%,1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, or 10%concentration. The optimal fixation concentration can be determinedempirically and can differ based on the type of biological sample to befixed (e.g., cells, tissue, type of tissue), the fixation reagent (e.g.,formaldehyde, paraformaldehyde, glutaraldehyde), the fixation method(e.g., drop fixation, on-slide fixation, or cardiac perfusion fixation).In some embodiments, the optimal fixation concentration for fixation byimmersion or on-slide fixation is 0.5%, 1%, or 2% paraformaldehydesolution. In some embodiments, the optimal fixation concentration forfixation by cardiac perfusion is 0.5%, 1%, 2%, or 4% paraformaldehydesolution. Typically, a suitable concentration of glutaraldehyde for useas a fixation reagent is 0.1 to 1%.

In some embodiments, fixing a biological sample using an aldehydeincludes incubating the biological sample with the aldehyde solution.The biological sample can be incubated with the aldehyde solution forbetween about 4° C. and 40° C. Fixing the biological sample can compriseimmersing the biological sample in an aldehyde solution of anytemperature range therein (e.g., 10° C. and 40° C., 15° C. and 40° C.,20° C. and 40° C., 15° C. and 35° C., 15° C. and 30° C., 20° C. and 35°C., 20° C., and 30° C.). In some embodiments, fixing the biologicalsample can comprise immersing the biological sample in an aldehyde atroom temperature.

Aldehyde fixation can be combined with other tissue preservationmethods. For example, aldehyde fixation can be combined with freshfrozen preservation of tissues. In this case, fresh tissue can beisolated from a subject and/or biological sample and flash frozen byimmersing the biological sample in isopentane chilled in liquidnitrogen. Once flash frozen, the biological sample can be moved to apetri dish or other support and covered with OCT embedding medium. Oncethe sample and OCT are frozen, the biological sample can be transferredto a cryostat for tissue sectioning. The biological sample can besliced/sectioned according to the methods described in SectionI(d)(ii)(1)-(2), and the sections can be placed on a slide (e.g., aspatially-barcoded array slide) and warmed in order to promotesample-slide adherence. The sample-mounted slides can be stored (e.g.,at −80° C.) until use. When ready for use, the sample-mounted slides areremoved from storage and allowed to dry at 37° C. for 1 minute. Thebiological sample can then be fixed in 2% paraformaldehyde under any ofthe conditions described above. For example, the sample can be incubatedin room temperature 2% paraformaldehyde for 10 minutes. After aldehydefixation, the biological sample can be subject to any of the staining orspatial analysis workflows described herein.

Aldehyde fixation of biological samples may require adjusting otherparameters or workflows described herein. For example, aldehyde fixationused with spatial analysis workflows may require longer permeabilizationperiods, additional permeabilization reagents, or higherpermeabilization reagent concentrations in order to liberate biologicalanalytes (e.g., mRNA) from a cross-linked biological sample for use inspatial analysis workflows described herein.

In some embodiments, the biological sample is de-crosslinked afterfixing the biological sample. In some embodiments, the biological sampleis de-crosslinked after both fixing the biological sample and placingthe biological sample on the substrate. In some embodiments, afterfixing the biological sample the biological sample is contacted with anagent for reversing formaldehyde fixation. In some embodiments, thebiological sample is digested after fixing the biological sample. Insome embodiments, the biological sample is digested after both fixingthe biological sample and placing the biological sample on thesubstrate. In some embodiments, the biological sample is digested withone or more digestive enzymes (e.g., any of the digestive enzymesdescribed herein).

(ii) Cardiac-Perfused Paraformaldehyde Fixation

An aldehyde can be used in cardiac perfusion to fix any biologicalsample described herein, e.g., any of the biological samples describedin Section I(d)(i). Non-limiting examples of aldehydes that can be usedin cardiac perfusion include formaldehyde, paraformaldehyde,glutaraldehyde, and combinations thereof. Cardiac-perfused fixation canbe especially helpful for preparing biological tissues forimmunohistochemical and immunofluorescent applications, as cardiacperfusion removes blood from a biological sample, which can contributeto staining artifacts and non-specific staining in these applications.

Tissues can be preserved and prepared for spatial analysis, wherein thetissues have been preserved by cardiac perfusion. For example, a mousebrain can be preserved by anesthetizing a mouse, perfusing with 50 mL ofchilled 1×PBS, followed by perfusing with 50 mL of chilled 4%paraformaldehyde (made with 1×PBS). The tissues of interest arecollected and placed in 4% paraformaldehyde overnight at 4° C. Thetissues can then be cryoprotected by incubating the tissues in 10%sucrose (made with 1×PBS) in 4° C. for 1 hour, followed by 20% sucrose(made in 1×PBS) in 4° C. for 1 hour, followed by 30% sucrose (made in1×PBS) in 4° C. overnight. After cryoprotecting the tissues, the tissuesare removed from the 30% sucrose solution and OCT embedded beforefreezing the tissue. Once completely frozen, the tissue sample can thenbe sectioned using a cryostat, and the resulting sections can be placedon a slide and warmed to promote sample adherence to the slide. Themounted tissue samples can be stored at −80° C. until ready for use. Insome embodiments, the biological sample can be cryoprotected in asucrose solution. In some embodiments, the sucrose solution can be abouta 3% to about a 40% sucrose solution. In some embodiments, the sucrosesolution can be about a 5% to about a 35% sucrose solution. In someembodiments, the sucrose solution can be about a 10% to about a 30%sucrose solution. In some embodiments, the sucrose solution can be abouta 15% to about a 25% sucrose solution. In some embodiments, the sucrosesolution can be about a 20% sucrose solution. In some embodiments, thesucrose solution can be about a 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,and about 40% sucrose solution.

In some embodiments, the biological sample can be cryoprotected in asucrose solution for about 5 minutes to about 90 minutes. In someembodiments, the biological sample can be cryoprotected in a sucrosesolution for about 10 minutes to about 85 minutes. In some embodiments,the biological sample can be cryoprotected in a sucrose solution forabout 15 minutes to about 80 minutes. In some embodiments, thebiological sample can be cryoprotected in a sucrose solution for about20 minutes to about 75 minutes. In some embodiments, the biologicalsample can be cryoprotected in a sucrose solution for about 25 minutesto about 70 minutes. In some embodiments, the biological sample can becryoprotected in a sucrose solution for about 30 minutes to about 65minutes. In some embodiments, the biological sample can be cryoprotectedin a sucrose solution for about 35 minutes to about 60 minutes. In someembodiments, the biological sample can be cryoprotected in a sucrosesolution for about 40 minutes to about 55 minutes. In some embodiments,the biological sample can be cryoprotected in a sucrose solution forabout 45 minutes to about 50 minutes. In some embodiments, contacting(e.g., cryopreserving) the biological sample with a sucrose solutionincludes contacting for about 1 hour to about 24 hours. In someembodiments, contacting the biological sample with a sucrose solutionincludes contacting for about 2 hours to about 23 hours. In someembodiments, contacting the biological sample with a sucrose solutionincludes contacting for about 3 hours to about 22 hours. In someembodiments, contacting the biological sample with a sucrose solutionincludes contacting for about 4 hours to about 21 hours. In someembodiments, contacting the biological sample with a sucrose solutionincludes contacting for about 5 hours to about 20 hours. In someembodiments, contacting (e.g., cryopreserving) the biological samplewith a sucrose solution includes contacting for about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours about 6 hours, about7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, and about 24 hours.

When ready for use, the mounted tissue samples are removed from storage,allowed to dry at 37° C. for 1 minute, and then placed in cold methanolat −20° C. for 5 minutes. After removing the tissue sample from themethanol, the tissue can be stained using any of theimmunohistochemistry or immunofluorescence workflows described herein.After performing the selected staining workflow, including imaging thestained tissue sample, the tissue sample can be further subjected toadditional workflows described herein (e.g., spatial analysisworkflows).

Cardiac-perfused fixation of biological samples may require adjustingother parameters or workflows described herein. For example,cardiac-perfusion fixation used with spatial analysis workflows mayrequire longer permeabilization periods, additional permeabilizationreagents, or higher permeabilization reagent concentrations in order toliberate biological analytes from a cross-linked biological sample.

In some embodiments, cardiac-perfused fixation of biological tissues canbe assessed by the spatial analysis workflows described herein. In someembodiments, spatial analysis can include spatial clustering of capturedgenetic transcripts. In some embodiments, the spatial clustering ofcaptured genetic transcripts that occurs in cardiac-perfused biologicaltissues is similar to control non-cardiac-perfused biological tissues.In some embodiments, the spatial clustering of captured genetictranscripts yields similar patters to control non-cardiac-perfusedbiological tissues.

(d) Immunohistochemistry (IHC)/Immunofluorescence (IF)

Both aldehyde and methanol fixation can be performed with the IHC and IFstaining protocols described herein. Additionally, aldehyde and methanolfixation can be performed on any of the spatial arrays provided herein.

In some embodiments, a substrate can be homogenously covered by aplurality of capture probes. In some embodiments, IHC and/or IFprotocols can be performed on a biological sample on a spatial array,wherein after the IHC and/or IF protocol is completed, the biologicalsample is treated to release analytes for capture by the spatial array.In some embodiments, an antibody of the IHC/IF protocol detects anextracellular protein (e.g., CD8, CD3). In some embodiments, theantibody of the IHC/IF protocol detects a cell surface protein. In someembodiments, the antibody of the IHC/IF protocol detects anintracellular protein (e.g., GFAP, NeuN). In some embodiments, theantibody of the IHC/IF protocol detects a nuclear protein. In someembodiments, IHC and/or IF protocols can be performed using a detectablelabel (e.g., radioisotopes, fluorophores, chemiluminescent compounds,bioluminescent compounds, and dyes, including any of the non-limitingexamples of detectable labels described in Section I(b)(xvi)). In someembodiments, a detectable label can be a fluorophore. In someembodiments, the biological sample can be imaged according to methodsdescribed herein or known in the art.

In some embodiments, IHC and/or IF protocols can be modified in order tooptimize spatial analysis. In some embodiments, a biological sample isblocked longer for a longer period of time when a biological sampleundergoes both IF protocols and spatial analysis than when thebiological sample undergoes IHC protocols and spatial analysis. In someembodiments, a biological sample requires a longer permeabilization timefor spatial analysis when a biological sample is fixed on a slide thanwhen the biological sample is not fixed on a slide. In some embodiments,ribonucleoside vanadyl complexes (RVC) are added to blocking and/orwashing buffers to help prevent degradation. In some embodiments, thebiological sample is de-coverslipped and the biological sample is notsubjected to an ethanol rinse and/or a drying step.

In some embodiments after the biological sample is imaged, thebiological sample can be processed and analyzed according to the spatialanalysis workflows described herein. In some embodiments, the IHC and/orIF antibodies are removed from the biological sample before performingthe spatial analysis workflows. In other embodiments, the IHC and/or IFantibodies are not removed from the biological sample before performingthe spatial analysis workflows.

(i) Immunohistochemistry

Immunohistochemistry is a technique used to examine protein expression,distribution, localization, and activation in situ. In some embodiments,immunohistochemistry uses an antibody conjugated to an enzyme to producea color-producing reaction. Immunohistochemistry can be used inidentifying abnormal cells, or to identify the distribution ofbiomarkers and differentially expressed proteins in different parts of abiological tissue. Immunohistochemistry can be used to identify a regionof interest for further processing.

Additionally, in some embodiments, staining the biological samplecomprises immersing the biological sample in hematoxylin for about 1minute to about 20 minutes. Staining a biological sample can be achievedby immersing the biological sample in hematoxylin for any range of timeperiods therein (e.g., 1 minute to 20 minutes, 1 minute to 19 minutes, 1minute to 18 minutes, 1 minute to 17 minutes, 1 minute to 16 minutes, 1minute to 15 minutes, 1 minute to 14 minutes, 1 minute to 13 minutes, 1minute to 12 minutes, 1 minute to 11 minutes, 1 minute to 10 minutes, 2minutes to 20 minutes, 3 minutes to 20 minutes, 4 minutes to 20 minutes,5 minutes to 20 minutes, 6 minutes to 20 minutes, 7 minutes to 20minutes, 6 minutes to 8 minutes, 5 minutes to 9 minutes, 4 minute to 10minutes, 3 minutes to 11 minutes, 2 minutes to 12 minutes or any subrange within these ranges). In some embodiments, staining comprisesimmersing the biological sample in hematoxylin for about a specific timeperiod (e.g., about 2 minutes, about 3 minutes, about 4 minutes, about 5minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13minutes, about 14 minutes, about 15 minutes). In some embodiments,staining the biological sample can comprise immersing the biologicalsample in hematoxylin for 7 minutes.

In some embodiments, staining a biological sample using hematoxylinincludes incubating the biological sample. The biological sample can beincubated between about at 4° C. and 40° C. Staining the biologicalsample can comprise immersing the biological sample in hematoxylin ofany temperature range therein (e.g., 10° C. and 40° C., 15° C. and 40°C., 20° C. and 40° C., 15° C. and 35° C., 15° C. and 30° C., 20° C. and35° C., 20° C. and 30° C.). In some embodiments, staining comprisesimmersing the biological sample in a primary antibody at an approximatetemperature (e.g., about 4° C., about 5° C., about 10° C., about 15° C.,about 20° C., about 25° C., about 30° C., about 35° C., about 40° C.).In some embodiments, staining the biological sample can compriseimmersing the biological sample in hematoxylin at room temperature.

Additionally, in some embodiments, staining the biological samplecomprises immersing the biological sample in eosin or eosin mix forabout 1 second to about 2 minutes. Staining a biological sample can beachieved by immersing the biological sample in eosin or eosin mix forany range of time periods therein (e.g., 10 seconds to 110 seconds, 20seconds to 100 seconds, 30 seconds to 90 seconds, 40 seconds to 80seconds, 50 seconds to 70 seconds, or any sub range within theseranges). In some embodiments, staining comprises immersing thebiological sample in eosin or eosin mix for about a specific time period(e.g., about 10 seconds, about 20 seconds, about 30 seconds, about 40seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 80seconds, about 90 seconds, about 100 seconds, about 110 seconds, about120 seconds). In some embodiments, staining the biological sample cancomprise immersing the biological sample in eosin or eosin mix for 1minute.

In some embodiments, staining a biological sample using eosin or eosinmix includes incubating the biological sample. The biological sample canbe incubated between about at 4° C. and 40° C. Staining the biologicalsample can comprise immersing the biological sample in eosin or eosinmix of any temperature range therein (e.g., 10° C. and 40° C., 15° C.and 40° C., 20° C. and 40° C., 15° C. and 35° C., 15° C. and 30° C., 20°C. and 35° C., 20° C. and 30° C.). In some embodiments, stainingcomprises immersing the biological sample in a primary antibody at anapproximate temperature (e.g., about 5° C., about 10° C., about 15° C.,about 20° C., about 25° C., about 30° C., about 35° C., about 40° C.).In some embodiments, staining the biological sample can compriseimmersing the biological sample in eosin or eosin mix at roomtemperature.

(ii) Immunofluorescence

Immunofluorescence is a technique used to examine protein expression,distribution, localization, and activation in situ. In some embodiments,immunofluorescence uses the specificity of fluorescently labeledantibodies or probes that bind to specific target analytes (e.g., withinor on a cell surface), and allows for the examination of thedistribution of the target analytes through the tissue sample. In someembodiments, immunofluorescence staining methods can be divided into twocategories, direct and indirect methods. In some embodiments, in thedirect staining method, a fluorophore is conjugated to an antibody(e.g., “the primary antibody”) which is capable of binding directly tothe target analyte of interest (e.g., cell surface or intracellulartarget). In some embodiments, in the indirect staining method, theprimary antibody binding to the target analyte is visualized by thebinding of a fluorescently labeled second-step antibody, wherein thesecond-step antibody is capable of binding to the primary antibody. Insome embodiments, the second-step antibody is an anti-immunoglobulinantibody.

In some embodiments, immunofluorescent imaging is performed and/ordetected individually for each analyte of interest. In some embodiments,immunofluorescent imaging can simultaneously detect one or more targetproteins in the same cell or tissue (e.g., multiplexed analytedetection). In some embodiments, multiplexing (e.g., simultaneouslydetecting multiple analytes) allows for examination of spatialarrangement of analytes of interest (e.g., proteins, DNA, RNA) as wellas analyte interaction and co-localization thereby facilitatingsimultaneous analysis of multiple tissue markers. Proper concentrationsof primary and secondary antibody can be determined by running anantibody dilution prior to the experiment/workflow.

Additionally, in some embodiments, staining the biological samplecomprises immersing the biological sample in a primary antibody forabout 1 minute to about 60 minutes. Staining a biological sample can beachieved by immersing the biological sample in a primary antibody forany range of time periods therein (e.g., 1 minute to 50 minutes, 1minute to 40 minutes, 1 minute to 30 minutes, 1 minute to 20 minutes, 10minutes to 60 minutes, 20 minute to 60 minutes, 30 minutes to 60minutes, 40 minutes to 60 minutes, 10 minutes to 60 minutes, 20 minutesto 50 minutes, 20 minutes to 40 minutes, or any sub range within theseranges). In some embodiments, staining comprises immersing thebiological sample in a primary antibody for about a specific time period(e.g., about 10 minutes, about 15 minutes, about 20 minutes, about 25minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45minutes, about 50 minutes, about 55 minutes, about 60 minutes).

In some embodiments, staining a biological sample using an antibodyincludes incubating the biological sample. The biological sample can beincubated between about at 4° C. and 40° C. Staining the biologicalsample can comprise immersing the biological sample in an antibody(e.g., primary or secondary antibody) of any temperature range therein(e.g., 10° C. and 40° C., 15° C. and 40° C., 20° C. and 40° C., 15° C.and 35° C., 15° C. and 30° C., 20° C. and 35° C., 20° C. and 30° C.). Insome embodiments, staining comprises immersing the biological sample inan antibody (e.g., primary or secondary antibody) at an approximatetemperature (e.g., about 4° C., about 5° C., about 10° C., about 15° C.,about 20° C., about 25° C., about 30° C., about 35° C., about 40° C.).In some embodiments, staining the biological sample can compriseimmersing the biological sample in the primary antibody at roomtemperature.

Further, in some embodiments, staining the biological sample comprisesimmersing the biological sample in a secondary antibody for about 1minute to about 60 minutes. Staining a biological sample can be achievedby immersing the biological sample in a secondary antibody for any rangeof time periods therein (e.g., 1 minute to 50 minutes, 1 minute to 40minutes, 1 minute to 30 minutes, 1 minute to 20 minutes, 10 minutes to60 minutes, 20 minute to 60 minutes, 30 minutes to 60 minutes, 40minutes to 60 minutes, 10 minutes to 60 minutes, 20 minutes to 50minutes, 20 minutes to 40 minutes, or any sub range within theseranges). In some embodiments, staining comprises immersing thebiological sample in a secondary antibody for about a specific timeperiod (e.g., about 10 minutes, about 15 minutes, about 20 minutes,about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes,about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes).

XII. Methods for Spatial Proteomics and Genomics

In some cases, it may be beneficial to combine one or more ofimmunofluorescence, spatial analysis of proteins, and/or spatial geneexpression analysis. Provided herein are methods of performing suchcombination.

Provided herein are methods for analysis of protein expression (e.g.,spatial proteomics) in a biological sample. In some cases, proteinexpression may be analyzed on its own, or it may be analyzed togetherwith transcriptomics. In some cases, provided herein are methods forpreparing a sample for analysis of protein expression (e.g., spatialproteomics).

Accordingly, provided herein is a method for preparing a biologicalsample for spatial proteomic analysis, the method including (a) placingthe biological sample on a substrate, wherein the substrate comprises aplurality of capture probes, where a capture probe of the plurality ofcapture probes comprises a spatial barcode and a capture domain; (b)contacting the biological sample with a protein blocking buffer; and (c)contacting the biological sample with a plurality of analyte captureagents, wherein an analyte capture agent of the plurality of analytecapture agents comprises an (i) analyte binding moiety that bindsspecifically to an analyte from the biological sample, (i) an analytebinding moiety barcode, and (iii) an analyte capture sequence, whereinthe analyte capture sequence binds specifically to the capture domain ofthe capture probe.

In some embodiments, the analyte from the biological sample can be aprotein. In some embodiments, the analyte from the biological sample canbe an extracellular protein. In some embodiments, the analyte from thebiological sample can be an intracellular protein. In some embodiments,the analyte from the biological sample can be a cell surface protein.

The steps can be carried out in any appropriate order.

In some embodiments, the method further includes (d) contacting thebiological sample with methanol for about 5 minutes to about 30 minutes(e.g., about 5 minutes to about 20 minutes or about 10 minutes) at aboutabout −30° C. to about 0° C. (e.g., about −25° C. to about −10° C., orabout −20° C.). For example, in some embodiments, step (d) is performedbefore step (a). In some embodiments, step (a) is performed before step(d). In some embodiments, step (d) is performed between step (a) andstep (b).

In some cases, the method can further include immunofluorescence imagingof the sample. This can be useful, in some cases, to compare to spatialproteomic analysis. Accordingly, in some embodiments, the method furtherincludes after step (c): (e) contacting the biological sample with afluorescently-labeled antibody. In some cases, the fluorescently-labeledantibody can bind to the same protein as the analyte binding moiety. Insome embodiments, the fluorescently-labeled antibody can bind to adifferent protein than the analyte binding moiety. In some embodiments,step (e) can be performed between steps (b) and (c). In someembodiments, step (e) is performed simultaneously with step (c). In someembodiments, step (e) is performed after step (c). Typically, aftercontacting the biological sample with a fluorescently-labeled antibody,the biological sample is imaged using fluorescence microscopy.Therefore, in some embodiments, the method further includes, after step(e): (f) performing fluorescence imaging of the biological sample. Insome embodiments, step (f) is performed before step (c). In someembodiments, step (f) is performed after step (c).

In some embodiments, the biological sample can be permeabilized (e.g.,using any of the permeabilization conditions herein). Therefore, in someembodiments, the method can further include, after step (c): (g)permeabilizing the biological sample. In some embodiments, thebiological sample can be permeabilized with one or more reagent(s),wherein the one or more reagents include a proteinase (e.g., ProteinaseK). Without being bound by any particular theory, it is believed thatthe proteinase can release the capture agent barcode domain from theanalyte capture agent. In some embodiments, step (g) is performed afterstep (f).

In some embodiments, the method further includes before step (f): (h)contacting the biological sample with a coverslip. For example, in someembodiments, step (h) can include contacting the biological sample witha coverslip in the presence of glycerol, and optionally an RNAseinhibitor.

The analyte binding moiety can be any appropriate analyte binding moiety(e.g., any of the analyte binding moieties described herein). Forexample, in some embodiments, the analyte binding moiety can be anantibody or antigen-binding fragment thereof.

The protein blocking buffer can have any appropriate components. In someembodiments, the protein blocking buffer can include a first RNAseinhibitor, a second RNAse inhibitor, an acid salt buffer, a non-specificblocking protein, and a surfactant. In some embodiments, the first RNAseinhibitor can include ribonucleotide vandyl complex (RVC) In someembodiments, the RVC is present in the protein blocking buffer in anamount of about 5 mM to about 50 mM (e.g., about 10 mM to about 40 mM,about 10 mM to about 30 mM, or about 20 mM). In some embodiments, theacid salt buffer can be present in about 1× to about 5× (e.g., about 2×to about 4× or about 3×) concentration. In some embodiments, the acidsalt buffer can be SSC (saline sodium citrate). In some embodiments, thenon-specific blocking protein can be present in an amount of about 1% toabout 5% (w/v) (e.g., about 1% to about 3% (w/v) or about 2% (w/v)). Insome embodiments, the non-specific blocking protein can be a serumalbumin (e.g., bovine serum albumin). In some embodiments, thesurfactant is a non-ionic surfactant. In some embodiments, thesurfactant is C₁₄H₂₂O(C₂H₄O)_(n) (e.g., TRITON™ X-100). In someembodiments, the surfactant can include TRITON™ X-100, NP-40,polysorbate 20, saponin, digitonin, LEUCOPERM™, or a combinationthereof. In some embodiments, the surfactant is present in the proteinblocking buffer in an amount of about 0.05% to about 0.2% (v/v) (e.g.,about 0.1% (w/v)). In some embodiments, the second RNAse inhibitor ispresent in the protein blocking buffer in an amount of about 0.5 U/μL toabout 5 U/μL (e.g., about 0.5 to about 3 U/μL, or about 1 U/μL). Thesecond RNAse inhibitor can be any appropriate RNAse inhibitor, forexample, Protector RNase inbhitor (Roche), RiboLock Rnase Inhibitor(Thermo Fisher Scientific), RNase Inhibitor (Cloned) (Ambion), RNase out(Invitrogen), or RNase Inhibitor (New England Biolabs). In someembodiments, the biological sample is contacted with the proteinblocking buffer for about 1 minute to about 10 minutes (e.g., about 2minutes to about 8 minutes, about 4 minutes to about 6 minutes, or about5 minutes). In some embodiments, the biological sample is contacted withthe protein blocking buffer at a temperature of about 1° C. to about 25°C. (e.g., about 2° C. to about 8° C., about 2° C. to about 6° C., about4° C., about 5° C. to about 15° C., about 15° C. to about 25° C., orabout 18° C. to about 22° C.).

In some embodiments the biological sample can be neurological tissue(e.g., human neurological tissue).

The capture probe can have any appropriate additional components, suchas any of the components described herein. In some embodiments, thecapture probe can include DNA. In addition, it will be understood that acapture probe or a plurality of capture probes may have differentcapture domains, for example, to capture different types of analytes. Insome embodiments, the capture domain can include a poly(T) sequence. Insome embodiments, the analyte capture sequence includes a poly(A)sequence, e.g., to be captured by the poly(T) sequence. In someembodiments, the analyte capture sequence does not include a poly(A)sequence. In some embodiments, the capture domain does not comprise apoly(T) sequence. In some such embodiments, the capture domain may havean alternate sequence binds specifically to the analyte capturesequence.

Also provided herein is a method of determining a location of a proteinanalyte in a biological sample, the method including: (a) preparing abiological sample according to any of the methods described herein; and(b) determining (i) a sequence corresponding to the capture agentbarcode domain or a complement thereof, and (ii) a sequencecorresponding to the spatial barcode of the capture probe or acomplement thereof, and using the determined sequences of (i) and (ii)to determine the location of the protein in the biological sample.

Also provided herein is a method of determining a location of a proteinanalyte and a nucleic acid analyte in a biological sample, the methodincluding (1) preparing a biological sample according to any of themethods provided herein, where: preparing the biological samplecomprises permeabilizing the biological sample, the analyte bindingmoiety binds specifically to the protein analyte, and the substratecomprises a second capture probe of the plurality of capture probes, thesecond capture probe comprising a spatial barcode and a capture domain,wherein the capture domain of the second capture probe bindsspecifically to the nucleic acid analyte; (2) determining (i) a sequencecorresponding to the capture agent barcode domain or a complementthereof, and (ii) a sequence corresponding to the analyte binding moietybarcode or a complement thereof, and using the determined sequences of(i) and (ii) to determine the location of the protein analyte in thebiological sample; and (e) determining (i) a sequence corresponding tothe nucleic acid analyte or a complement thereof, and (ii) a sequencecorresponding to the spatial barcode of the second capture probe or acomplement thereof, and using the determined sequences of (i) and (ii)to determine the location of the nucleic acid analyte in the biologicalsample.

The steps can be performed in any appropriate order. In someembodiments, step (b) is performed before step (c). In some embodiments,step (b) is performed simultaneously with step (c). In some embodiments,step (d) is performed before step (e). In some embodiments, step (d) isperformed after step (e). In some embodiments, step (d) is performedsimultaneously with step (e).

Determining the sequences of (i) and (ii) can be performed using anyappropriate method. In some embodiments, determining of the sequences of(i) and (ii) comprises sequencing the sequences of (i) and (ii). In someembodiments, the sequencing is high throughput sequencing.

EXAMPLES Example 1. Method for Profiling a Biological Analyte in aPlurality of Cells, an Organism, or an Organoid

A biological sample (e.g., a plurality of cells, an organism, or anorganoid) is disposed upon a solid support comprising a plurality ofcapture probes. See FIG. 25. The capture probes of the solid supportindividually include a molecular barcode and a capture domain. Adistinct spatial position on the solid support comprises capture probesthat are distinct from other capture probes of the plurality of captureprobes. The cells of the biological sample are lysed such thatbiological analytes from the cells are released and bind to a captureprobe at the distinct spatial position of the solid support. The captureprobes/analytes are analyzed, and the biological analyte with amolecular barcode of the capture probe is correlated with the distinctspatial position of the solid support, thus profiling the biologicalanalyte as having been cell.

Example 2. Method for Profiling a Biological Analyte in a Plurality ofCells Using a Substrate Comprising Capture Antibodies

A plurality of cells is distributed onto a substrate comprising featureswith a plurality of capture probes and a plurality of captureantibodies. The capture probes of the solid support individuallycomprise a molecular barcode and a capture domain. A distinct spatialposition on the solid support comprises capture probes that are distinctfrom other capture probes of the plurality of capture probes. Cells ofthe plurality of cells bind to capture antibodies on the substrate. Thecells are lysed such that biological analytes from the cells arereleased and bind to a capture probe at the distinct spatial position ofthe solid support. The capture probes/analytes are analyzed, and thebiological analyte with a molecular barcode of the capture probe iscorrelated with the distinct spatial position of the solid support, thusprofiling the biological analyte as having been present in the cell.

Example 3. Method for Profiling a Biological Analyte in a Migrated Cell

A plurality of cells is allowed to migrate on a substrate having aplurality of capture probes and having channels that confine cellmigration to one dimension along the substrate. The capture probes ofthe solid support individually comprise a spatial barcode and a capturedomain. A distinct spatial position on the solid support comprisescapture probes that are distinct from other capture probes of theplurality of capture probes. The cells are lysed such that biologicalanalytes from the cells are released and bind to a capture probe at thedistinct spatial position of the solid support. The captureprobes/analytes are analyzed, and the biological analyte with amolecular barcode of the capture probe is correlated with the distinctspatial position of the solid support, thus profiling the biologicalanalyte as having been present in the cell.

Example 4. Method for Profiling a Biological Analyte in SubcellularFractions

A biological sample is placed on a slide, and the slide is contactedwith an array as described herein. A lysis solution that selectivelypermeabilizes the plasma membrane is added to the biological sample.Target biological analytes in the cytosol are released from thebiological sample and diffuse towards the array and are bound by captureprobes. The array is removed, and reverse transcription/barcoding can beperformed on the array. Additionally, the capture probes are collectedor second strand synthesis is performed for preparation of a library.The biological sample is then contacted with a second array and anucleus-specific lysis solution is added to the biological sample.Target biological analytes from the nuclei of cells in the biologicalsample are released and diffuse towards the second array and are boundby capture probes. The second array is removed, and reversetranscription/barcoding can be performed on the second array.Additionally, the capture probes from the second array are collected orsecond strand synthesis is performed for preparation of a library.

Example 5. Temporal Analysis of an Analyte in a Biological Sample

Live cells tissue sections are placed in a controlled environmentpermissive for cellular growth and/or maintenance, and/or to preventhypoxia. Barcoded arrays are placed on top of the tissues and imagedusing a microscope or other suitable instrument to register the relativeposition of the tissue to the barcoded array, optionally using opticallyencoded fiducial markers. An electric field is applied for a period oftime between the live biological sample and the barcoded array, suchthat biological analytes (e.g., DNA, RNA, proteins, metabolites, smallmolecules, lipids, and the like) are released and captured onto thespatially barcoded array, preserving their spatial information. Thearray is removed, and the molecular information therein is determined byperforming library construction for next generation sequencing.Sequencing is followed by computational analysis to correlate theexpression value with spatial barcode. These steps are repeated one ormore times to capture the analytes' spatial information over varioustime-points.

Example 6. Methods for Spatially Detecting One or More Proteins from aBiological Sample

In a non-limiting example, antibody staining is performed on a captureprobe array. FIG. 36 shows an exemplary illustration of fluorescentlylabeled antibodies interacting with analytes of a biological sample on acapture probe array. For example, a method of antibody staining caninclude: (a) providing a capture probe array, as described herein; (b)contacting a tissue section (e.g., mouse spleen tissue) with the arrayand drying the sectioned slides for 1 minute at 37° C.; (c) fixing thetissue section with either 2% formaldehyde at room temperature or withmethanol at −20° C. for 10 minutes; (d) rehydrating, blocking, andpermeabilizing the tissue sample with a blocking buffer (3×SSC, 2% BSA,0.1% Triton X, and 1 U/μl RNAse inhibitor) for 10 minutes at 4° C.; (e)staining the tissue section with fluorescent primary antibodies, forexample, the primary antibody is diluted 1:100 in an antibody dilutionbuffer (3×SSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAse inhibitor) for 30minutes at 4° C.; (f) removing the antibody by washing the sample 4times in 3×SSC, 2% BSA, 0.1% Triton X, and 1 U/μl RNAse inhibitor; (g)mounting and cover slipping the tissue sample in glycerol and 1 U/μlRNAse inhibitor; (h) imaging the tissue section to detect targetproteins within the tissue; (i) removing the coverslip and washing thetissue section in 3×SSC; and (j) performing any of the spatial workflowsdescribed herein. The steps of this method are depicted in FIG. 37.Performing any of the spatial workflows described herein can includedetermining the sequence of all or a portion of the spatial barcode or acomplement thereof, and all or a portion of the analyte binding moietybarcode, or complement thereof. In some embodiments of the example, theanalyte capture agent binds to the capture domain before detecting thelabel associated with the analyte capture agent. In some embodiments ofthe example, the analyte capture agent bind to the capture domain afterdetecting the label associated with the analyte capture agent.

The fluorescence level across the tissue can be imaged to produce animage wherein the image shows the fluorescently labeled antibodiesspecifically bind to the target proteins allowing for the examination ofdistribution of the target proteins throughout the tissue.

FIGS. 38 and 42 show exemplary staining using the method depicted aboveand in FIG. 37, wherein the antibody staining is performed on a captureprobe array. FIG. 38 shows tissue sections of mouse spleen fixed ineither 2% formaldehyde or 100% methanol. Mouse spleen contains red pulpcontaining stromal tissue, blood vessels, and macrophages, surroundingpockets of white pulp containing mostly immune cells, such as T cellsand B cells. In both the 2% formaldehyde and 100% methanol fixationconditions, CD3, a cell surface marker for T cells, can be seen in thepockets of white pulp, and CD29, a cell surface marker for stromalcells, can be seen in the red pulp portion of the spleen (FIG. 38). FIG.42 shows coronal sections of mouse brain fixed in either 2% formaldehydeor 100% methanol. Brain tissue is comprised mostly of neuronal and glialcells, organized into distinct compound structures, wherein the distinctcompound structures may be further comprised of layers or substructureswithin the compound structure. Particular substructures may be composedprimarily of neuronal bodies, or bundled axonal tracts. In these coronalsections, the CA1-CA3 regions of the hippocampus as well as the cerebralcortex can be clearly seen marked with NeuN, a common intracellularbiomarker for neurons. The dentate gyrus and cerebral cortex can bevisualized with DAPI, a common DNA stain. Additionally, GFAP (glialfibrillary acidic protein) stains glial cells intracellularly, includingastrocytes which contribute to the functioning of the blood-brainbarrier. FIG. 42 also demonstrates that different fixation protocols canaffect the data obtained from antibody staining. For example, with 100%methanol fixation (right) it is likely the NeuN antigen epitope has beenmasked, denatured, or the protein washed away via the permeabilizationeffects of methanol. The result is no NeuN antibody staining with 100%methanol fixation.

Example 7. Methods of Using Spatial Transcriptomics Analysis to IdentifyCell Type “Clusters”

In a non-limiting example, spatial analysis was performed on mousespleen (FIGS. 39-41) and brain coronal slices (FIGS. 43-45).

A t-SNE plot was generated which identified distinct cell typeclustering (FIGS. 39 and 43). The plot demonstrated the differentialexpression of genes captured using the spatial analysis methodsdescribed herein. The differential gene expression identified byclustering can be mapped back onto an image of the original biologicalsample in order to visualize the identity of the cells in the originaltissue sample (FIGS. 39 and 43). FIGS. 40 and 41 show exemplaryimmunofluorescence staining on the left, identifying the presence andlocation of CD3, CD29, and DAPI. The immunofluorescence staining isoverlaid with CD3 transcript counts (measured in UMI during spatialtranscriptome analysis) (right), demonstrating that specific analytescan be captured and barcoded according to the spatial workflowsdescribed herein. FIGS. 44 and 45 show exemplary immunofluorescencestaining of mouse brain (left) in three channels (NeuN; GFAP; DAPI). Theimmunofluorescence staining is overlaid with NeuN transcript counts(measured in UMI during spatial transcriptome analysis) (top) and GFAPUMI counts (bottom), demonstrating that specific analytes can becaptured and barcoded according to the spatial workflows describedherein.

Example 8. Methods for Blocking an Analyte Capture Sequence Binding to aCapture Probe

In a non-limiting example, non-specific antibody staining can be reducedby introducing a reversible blocking probe to the analyte captureagent(s), prior to applying the analyte capture agents to a tissuesample. FIG. 46 shows an exemplary illustration of an analyte capturesequence (a poly(A) sequence) blocked by a poly(T) sequence blockingprobe, inhibiting the fluorescently labeled antibody from interactingwith capture probes on a capture probe array. When the reversibleblocking probe is removed (e.g., by heating the analyte capture agent),the analyte capture sequence is free to interact with (e.g., hybridizeto) the capture domain of capture probes on the array.

A method of blocking an analyte capture sequence can includeDNA-barcoded antibodies including an analyte binding moiety (e.g.,antibody) and a capture agent barcode domain. The capture agent barcodedomain includes an analyte capture sequence, which could bind to thecapture probe capture domain prior to analyte capture by the analytebinding moiety. The analyte capture sequence binding to a capture probecan be blocked by hybridizing the analyte capture sequence (e.g., thepoly(A) 3′ tail of the capture agent barcode domain) with a blockingprobe. In this example, the blocking probes comprise an oligo (dT)sequence that can interact with the poly(A) tails of the analyte capturesequence (FIG. 46). Here, the analyte capture agent analyte capturesequence can be blocked before applying the analyte capture agents tothe tissue sample. Additionally, excess blocking probes can be providedin a sample hydration solution (e.g., a permeabilization solutionapplied to the tissue sample).

In some embodiments, the analyte capture sequence can be reversiblyblocked with a blocking probe prior to applying the analyte captureagents to the tissue sample, and when treated with a nuclease (e.g.,RNAse H) the blocking probe is digested, allowing the analyte capturesequence to interact with capture probes on the substrate. See, forexample, FIG. 47.

FIG. 47 shows the performance of an oligo (dT) blocking probe undervarious conditions. The effectiveness of the oligo (dT) blocking probesvaried as a function of the length of the blocking probe and thetemperature of the array during the antibody staining protocol. Here,the above experiment was performed with oligo (dT) blocking probes ofvarying lengths at 4° C. and 37° C. FIG. 48 indicates analyte captureagents were blocked from binding to the capture probes at bothtemperatures, although shorter blocking probes were more effective underlower temperatures.

Example 9. Methods for Using a Spatially-Tagged Analyte Capture Agent ina Biological Sample

In a non-limiting example, DNA-barcoded antibodies are used to detectproteins in a biological sample. For example, a method of detectingproteins within a tissue sample using DNA-barcoded antibodies caninclude: (a) providing a capture probe array, wherein the capture probesinclude a spatial barcode and a capture domain; (b) contacting thesubstrate with a tissue sample (e.g., mouse spleen tissue) and dryingthe sectioned slides for 1 minute at 37° C.; (c) fixing the tissuesample with either 2% formaldehyde at room temperature or with methanolat −20° C. for 10 minutes; (d) rehydrating, blocking and permeabilizingthe tissue sample with 3×SSC, 2% BSA, 0.1% Triton X, and 1 U/μl RNAseinhibitor for 10 minutes at 4° C.; (e) staining the tissue sample withfluorescent primary antibodies and a pool of DNA-barcoded antibodies in3×SSC, 2% BSA, 0.1% Triton X, and 1 U/μl RNAse inhibitor for 30 minutesat 4° C.; (f) imaging the tissue sample to spatially detect targetproteins (e.g., CD29, CD3) within the tissue usingfluorescently-labelled and DNA-barcoded antibodies; (g) treating thetissue sample with a protease to permeabilize the tissue and release theantibody oligos; and (h) performing spatial transcriptomic analysis toidentify the location of the target protein within the tissue sample.The steps of this method are depicted in FIG. 49.

The DNA-barcoded antibodies can include an analyte binding moiety (e.g.,antibody) and a capture agent barcode domain. The antibodies interactwith the target protein of the biological sample, and the capture agentbarcode domains interact with the capture probes on the substrate. Thefluorescence level from the primary antibodies interacting with theproteins of the biological sample is imaged in step (f), and thespatially-tagged analyte capture agents associated with the captureprobes are used to identify the location of the target protein withinthe biological sample (FIG. 50). In some embodiments, non-specificantibody staining can be reduced by introducing a blocking probe to theanalyte capture agent(s), prior to applying the analyte capture agentsto a tissue sample, as described in Example 3.

In some embodiments, detecting and identifying the location of a targetprotein can be performed individually for each analyte of interest. Insome embodiments, multiple proteins can be detected and spatiallyprofiled simultaneously within the same tissue sample. In someembodiments, multiplexing (e.g., simultaneously detecting multiplemarkers) allows for examination of the spatial arrangement of analytesof interest (e.g., proteins, DNA, RNA) as well as analyte interactionand co-localization thereby facilitating simultaneous analysis ofmultiple tissue markers.

FIG. 50 shows exemplary imaging results using the method depicted aboveand in FIG. 49, wherein the left image shows a tissue section of mousespleen with fluorescent antibodies bound to CD29. The images on theright show the location of target proteins, CD29, CD3, F4/80, and CD169within the tissue sample using the spatially-tagged analyte captureagents associated with the surface capture probes on the substrate. Inparticular, the method above was performed by fixing the tissue samplewith 2% formaldehyde and permeabilizing with proteinase K.

Example 10. Methods for Using Spatially-Tagged Analyte Capture Agents toDetect Multiple Target Proteins by Introducing Antibodies Linked toCapture Agents and Molecular Identifiers in a Biological Sample

In a non-limiting example, multiple pluralities of DNA-barcodedantibodies can be used to simultaneously detect multiple target proteins(e.g., multiplexing) within a biological sample. For example, a methodof detecting multiple target proteins within a biological sample caninclude the method described in Example 4, wherein two or morepluralities of analyte capture agents bind to two or more pluralities ofanalytes. Each analyte species is associated with a spatially-taggedanalyte capture agent plurality, wherein each spatially-tagged analytecapture agent plurality possesses a barcode unique to the analyte.Multiple analytes can be detected and analyzed at the same time bydetermining the analyte binding moiety barcode that can be determinedtogether with, or separately from the spatial transcriptome analysisusing a sequencing technology, as described elsewhere herein. In otherembodiments, antibody-barcodes can be determined by fluorescent in situhybridization or in situ sequencing approaches, as described elsewhereherein or known in the art.

For example, FIG. 51 shows exemplary multiplexed DNA-barcoded andfluorescent antibody staining and sequencing results using the methoddepicted above, wherein the left immunofluorescent image shows tissuesections of mouse spleen with fluorescent and DNA-barcoded antibodiesbound to CD29 and CD4. CD29 (Integrin beta 1) is a cell surface markerexpressed in many stromal cells and can be seen in the red pulp portionof the spleen, while CD4 is a cell surface marker for T cell subsets andcan be seen in the pockets of white pulp of the spleen. The images onthe right show the location of the antibody barcodes recognizing targetproteins, CD29, CD3, CD4, CD8, CD19, B220, F4/80, and CD169 within thetissue sample using the multiple spatially-tagged analyte captureagents. Each spatially-tagged analyte capture agent plurality possess ananalyte binding moiety barcode unique to that plurality. FIG. 51 showsthat CD3, CD4, and CD8, all cell surface markers for T cells, are seento be located in the pockets of white pulp of the spleen. CD19, a cellsurface marker for B cells, CD29, a cell surface marker for stromalcells, and F4/80 and CD169, both markers for macrophage cells, can beseen within the red pulp and white pulp respectively of the spleentissue. The data of FIG. 51 indicates that protein detection usingDNA-barcoded antibodies can be used for simultaneously identifyingspatial locations of multiple (e.g., two or more, three or more) targetproteins within a tissue sample.

Example 11. Methods for Tissue-Optimization to Test Staining withFluorescently Labelled Antibodies and Optimal PermeabilizationConditions

In a non-limiting example, antibody staining and tissue permeabilizationare optimized before performing the methods disclosed herein. Oneexample of a method of optimizing antibody staining and imaging caninclude: (a) providing a capture probe array, as described herein; (b)contacting the substrate with a tissue sample (˜10 μm tissue section)and drying the sectioned slides for 1 minute at 37° C.; (c) fixing thetissue sample with either 1% formaldehyde for 10 minutes at roomtemperature or with 100% methanol at −20° C. for 30 minutes or longer;(d) mounting slides into slide cassettes without drying the slides; (e)rehydrating and blocking the tissue sample with 3×SSC, 0.1% Triton X, 2%BSA, 1 U/μl RNAse inhibitor, and 10 mM ribonucleoside vanadyl complexfor 5 minutes at 4° C.; (f) removing the blocking buffer; (g) stainingthe tissue sample with fluorescent antibodies and blocking oligos in3×SSC, 0.1% Triton X, 2% BSA, and 2 U/μl RNAse inhibitor for 30 minutesat 4° C. protected from light; (h) washing the tissue sample with 3×SSC,0.1% Triton X, 2% BSA, 1 U/μl RNAse inhibitor, and 10 mM ribonucleosidevanadyl complex for 1 minute at room temperature; (i) dipping the tissuesample 20 times in 3×SSC; (j) mount tissue in mounting medium (90%glycerol and RNAse Inhibitor); and (k) imaging the tissue sample toevaluate the quality of fluorescent antibody staining.

Additionally, a method of optimizing permeabilization conditions for abiological sample can include: (a) providing a capture probe array, asdescribed herein; (b) contacting the substrate with a tissue sample(e.g., —10 μm tissue section) and drying the sectioned slides for 1minute at 37° C.; (c) fixing the tissue sample with either 1%formaldehyde for 10 minutes at room temperature or with 100% methanol at−20° C. for 30 minutes or longer; (d) mounting slides into slidecassettes without drying the slides; (e) rehydrating and blocking thetissue sample with 3×SSC, 0.1% Triton X, 2% BSA, 1 U/μl RNAse inhibitor,and 10 mM ribonucleoside vanadyl complex for 5 minutes at 4° C.; (f)removing the blocking buffer; (g) staining the tissue sample withfluorescent antibodies and blocking oligos in 3×SSC, 0.1% Triton X, 2%BSA, and 2 U/μl RNAse inhibitor for 30 minutes at 4° C. protected fromlight; (h) washing the tissue sample with 3×SSC, 0.1% Triton X, 2% BSA,1 U/μl RNAse inhibitor, and 10 mM ribonucleoside vanadyl complex for 1minute at room temperature; (i) removing washing buffer from tissuesample; (j) incubating the tissue sample with a permeabilization mix oftissue removal enzyme, 3×SSC, and 10% SDS for 3, 6, 9, 12, or 18 minutesat 37° C. to permeabilize the tissue and release the antibodies; (k)removing the permeabilization mix after the incubation period andwashing twice with 0.1×SSC; and (l) performing a reverse transcriptionprotocol and evaluating the optimal permeabilization conditions fordifferent permeabilization times. Different samples of tissue can betreated with different permeabilization times (3, 6, 9, 12, or 18minutes) to identify the optimal permeabilization conditions for thatparticular sample type.

Example 12. Methods of Library Preparation for Protein Detection

In a non-limiting example, library preparation for protein detectionrequires different buffers and reagents compared to standard librarypreparation protocol. For example, a 2× Blocking Buffer comprising6×SSC, 0.2% Triton X, 4% BSA, 0.2 μg/μl sheared salmon sperm, 2 U/μlRNAse Inhibitor, and a 1× Blocking Buffer comprising 3×SSC, 0.1% TritonX, 2% BSA, 0.1 μg/μl sheared salmon sperm, 1 U/μl RNAse Inhibitor, canbe prepared beforehand. Additionally, an Antibody Staining Mixcomprising 1× Blocking Buffer, 20 μM Blocking Oligos (dT25), 1 U/μlRNAse Inhibitor, fluorescent antibodies, and Totalseq A Antibody poolcan be prepared beforehand as well as a Washing Buffer comprising 1×Blocking Buffer and 10 mM Ribonucleoside Vanadyl Complex. Finally, theMounting Medium for the slide can comprise 90% glycerol and RNAseInhibitor. These buffers and reagents can be used in the methods belowfor protein/antibody detection in combination with the oligonucleotideworkflows described herein.

In one example, a method for preparing a TotalSeqA (BioLegend) antibodypanel can include: (a) pooling an appropriate amount of TotalSeqAantibodies to create a panel of interest; (b) preparing an AmiconUltra-0.5 50 kDa MWCO filter unit with 3×SSC; (c) adding the antibodypool to the filter and spinning the unit at 14,000 g for 5 minutes; (d)discarding the flow through and adding 3×SSC; (e) spinning the sample at14,000 g for 5 minutes; and (f) inverting the filter into a collectiontube and spinning the collection tube at 1,000 g for 2 minutes, therebyrecovering the antibody pool. In some embodiments, when pooling largenumbers of antibodies, the storage buffer comprises 3×SSC. In someembodiments, 1 μg/μl BSA and 0.06% sodium azide is added to therecovered antibody pool.

In one example, a method of library preparation for protein detectioncan include: (a) providing a capture probe array, as described herein;(b) contacting the substrate with a tissue sample (e.g., ˜10 μm tissuesection) and drying the sectioned slides for 1 minute at 37° C.; (c)fixing the tissue sample with either 1% formaldehyde for 10 minutes atroom temperature or with 100% methanol at −20° C. for 30 minutes orlonger; (d) mounting slides into slide cassettes without drying theslides; (e) rehydrating and blocking the tissue sample with 3×SSC, 0.1%Triton X, 2% BSA, 0.1 μg/μl sheared salmon sperm, 1 U/μl RNAseinhibitor, and 10 mM ribonucleoside vanadyl complex for 5 minutes at 4°C.; (f) removing blocking buffer from the tissue; (g) staining thetissue sample with fluorescent antibodies and blocking oligos in 3×SSC,0.1% Triton X, 2% BSA, and 2 U/μl RNAse inhibitor for 30 minutes at 4°C. protected from light; (h) washing the tissue sample 4 times with3×SSC, 0.1% Triton X, 2% BSA, 1 U/μl RNAse inhibitor, and 10 mMribonucleoside vanadyl complex for 1 minute at room temperature; (i)dipping tissue slide 20 times in 3×SSC; (j) imaging the tissue sample todetect visible antibodies (e.g., Cy3), wherein a fiducial frame isvisible on the slide; (k) washing the tissue with washing buffer andremoving the washing buffer; (l) incubating the tissue sample with aneven covering of tissue removal enzyme, 3×SSC, and 10% SDS at 37° C. topermeabilize the tissue and release the antibodies for an optimal amountof time, as determined in Example 6; (m) removing the tissue from thepermeabilization mix and washing twice with 0.1×SSC; and (n) performinga reverse transcription protocol according to methods described herein.

In another example, a method of library preparation for proteindetection with second strand synthesis can include: (a) providing acapture probe array, as described herein; (b) contacting the substratewith a tissue sample (e.g., —10 μm tissue section) and drying thesectioned slides for 1 minute at 37° C.; (c) fixing the tissue samplewith either 1% formaldehyde for 10 minutes at room temperature or with100% methanol at −20° C. for 30 minutes or longer; (d) mounting slidesinto slide cassettes without drying the slides; (e) rehydrating andblocking the tissue sample with 3×SSC, 0.1% Triton X, 2% BSA, 0.1 μg/μlsheared salmon sperm, 1 U/μl RNAse inhibitor, and 10 mM ribonucleosidevanadyl complex for 5 minutes at 4° C.; (f) removing blocking bufferfrom the tissue; (g) staining the tissue sample with fluorescentantibodies and blocking oligos in 3×SSC, 0.1% Triton X, 2% BSA, and 2U/μl RNAse inhibitor for 30 minutes at 4° C. protected from light; (h)washing the tissue sample 4 times with 3×SSC, 0.1% Triton X, 2% BSA, 1U/μl RNAse inhibitor, and 10 mM ribonucleoside vanadyl complex for 1minute at room temperature; (i) dipping tissue slide 20 times in 3×SSC;(j) imaging the tissue sample to detect visible antibodies, wherein afiducial frame (e.g. Cy3) is visible on the slide; washing the tissuewith washing buffer and removing the washing buffer; (l) treating thetissue sample with an even covering of tissue removal enzyme, 3×SSC, and10% SDS to permeabilize the tissue and release the antibodies for anoptimal amount of time, as determined in Example 6; (m) removing thetissue from the permeabilization mix and washing twice with 0.1×SSC; (n)performing a reverse transcription protocol; and (i) adding 2 μl ofadditive primer (200 μM) to a second strand synthesis mix and performsecond strand synthesis according to the methods described herein. Forexample, second strand synthesis can be performed by removing a reversetranscriptase master mix from the tissue; adding 0.08M KOH to thetissue; adding elution buffer to the tissue; removing elution bufferfrom the tissue; adding a second strand mix to the tissue, wherein thesecond strand mix comprises second strand reagent, second strand primer,second strand enzyme, and additive primers; subjecting the tissue to athermocycling program comprising 65° C. for second strand synthesisfollowed by a 4° C. hold. Other library preparation steps can becompleted as described herein.

In another example, a method of cDNA amplification and cleanup caninclude: (a) preparing a cDNA amplification mix on ice; (b) addingadditive primer and cDNA primers to the cDNA amplification mix toincrease yield of antibody-products; and (c) performing cDNAamplification as described herein. In some embodiments, additive primeris not added to qPCR to determine amplification cycles. In someembodiments, cDNA amplification is performed one additional cycle thanwhat was determined in the qPCR.

In another example, a method of cDNA and antibody-product size selectioncan include: (a) separating cDNA amplification antibody-products (e.g.,˜180 bp) and mRNA-derived cDNAs (e.g., >300 bp) by 0.7×SPRI, wherein thebead fraction contains mRNA derived cDNAs and supernatant contains ADTs;(b) adding 0.7×SPRI reagent to the cDNA reaction and incubating for 5minutes at room temperature; (c) placing the cDNA reaction on the magnethigh position for ˜1 minute until solution is clear; (d) transferringthe supernatant to a low-bind tube; and (e) performing cDNA cleanup andlibrary preparation with as described herein with beads. In someembodiments, the supernatant is transferred to a low-bind tube and usedto perform antibody-product cleanup. Other library preparation steps canbe completed as described herein.

In another example, a method of antibody-product cleanup can include:(a) purifying antibody-products from highly concentrated cDNAamplification primers by two rounds of 1.9×SPRI cleanups; (b) addingSPRI beads to supernatant to obtain a final SPRI to sample ratio of 1.9×and incubating 5 minutes at room temperature; (c) placing tube on magnetand waiting ˜2 minutes until solution is clear; (d) removing anddiscarding the supernatant; (e) adding 80% ethanol to the tube andremoving the ethanol wash; (f) resuspending beads in water; (g)performing another round of 1.9×SPRI purification by adding SPRI reagentdirectly onto resuspended beads and incubating 5 minutes at roomtemperature; (h) placing tube on magnet and wait ˜2 minutes untilsolution is clear; (i) removing and discarding the supernatant; (j)adding 80% Ethanol, without disturbing the pellet, letting stand for 30seconds and removing the ethanol wash; (k) repeating the ethanol wash;(l) airdrying the beads for 2 minutes and resuspending beads in water;and (m) placing tube on magnet and transferring clear supernatant into aPCR tube.

In another example, a method of antibody sequencing libraryamplification can include: (a) preparing a PCR reaction of purifiedADTs, wherein the PCR reaction comprises purified antibody-product,amplification mix, TruSeq small RNA RPIx primer, and SI-PCR primer; (b)cycling the PCR reaction: 95° C. for 3 minutes, 95° C. for 20 seconds,60° C. for 30 seconds, 72° C. for 20 seconds, 72° C. for 5 minutes, forapproximately 6-10 cycles; (c) purifying antibody PCT product by addingSPRI reagent to sample and incubating 5 minutes at room temperature; (d)placing tube on magnet high position and waiting ˜1 minute untilsolution is clear; (e) removing and discarding the supernatant; (f)adding 80% Ethanol to the tube for 30 seconds and removing the ethanolwash; (g) repeating the ethanol wash; (h) airdrying the beads for 2minutes and resuspending beads in water; (i) mixing the beads and waterand incubating at room temperature for 5 minutes; (j) placing tube onmagnet and transferring clear supernatant into a PCR tube; (k)quantifying the prepared antibody libraries by standard methodsdescribed herein (antibody libraries can be ˜180 bp; and (l) sequencingthe antibody libraries. Other library preparation steps can be completedas described herein.

Example 13. Exemplary Methanol Fixation Workflows with H&E Staining

In a non-limiting example, methanol fixation can be performed inconjunction with hematoxylin & eosin staining according to the followingworkflow (FIG. 54). Forty ml/slide of methanol can be pre-chilled to−20° C. in a centrifuge tube. A tissue slide adaptor plate, for examplean adaptor plate that will hold tissue slides that fits into athermocycler, can be placed on a thermal cycler set at 37° C. andallowed to equilibrate for 5 minutes. Thermocyclers oftentimes haveheated lids, for this example, heating the thermocycler lid is notrequired and the lid should not be used or closed at any time duringthis exemplary method. Tissue previously sectioned and placed on spatialarray slides can be removed from −80° C. and placed on dry ice in asealed container. Of note, a delay in transferring slides to dry ice mayresult in condensation, which may cause tissue damage and/or shifting oftissue sections on the array, such as the transfer to the dry ice shouldbe done as quickly as possible. The slide can be placed on thepre-equilibrated adapator plate (e.g., as many slides as the adaptorplate can accommodate, for example one, two or more slides) with theactive surface facing up and the slide can be incubated for 1 minute at37° C. More than one slide can potentially be processed at one time, inthat event the following methods can be followed for each slide.Following the 1 min incubation at 37° C., the slide can be carefullytaken out of the plate adaptor, any condensation wiped from the back ofthe slide (i.e., the side of the slide without tissue) and the slide canbe gently immersed in the pre-chilled methanol. For example, a slide canbe placed into a conical centrifuge tube that contains the pre-chilledmethanol wherein the whole slide is completely immersed in the methanolin the tube. To prevent methanol loss, the lid of the tube can bereplaced. The slide can be incubated in the pre-chilled methanol for 30minutes at −20° C. Following the methanol fixation for 30 minutes at−20° C. the slide can be stained if desired. For example, the methanolfixed tissues can be stained with haematoxylin and eosin (H&E) dyes forlocalization of nuclear and cellular structures.

FIG. 55 provides an example of an H&E staining workflow for tissues postfixation. Milli-Q water can be dispensed into four containers: 50 ml inone 50 ml centrifuge tube, 800 ml in three separate beakers. An Eosinmix can be prepared by combining 100 μl/slide of Eosin Y Solution (10×Genomics, Inc.) and 900 μl/slide of Tris-Acetic Acid Buffer (0.45 M, pH6.0) for a total volume of 1 ml. The slide can be removed from themethanol and the excess liquid wiped from the back of the slide withouttouching the tissue sections. Isopropanol (500 μl) can be added touniformly cover all tissue sections on the slide, and the tissuesections can be incubated for 1 minute at room temperature. Reagents arediscarded, for example, by draining and/or holding the slide at an anglewith the bottom edge in contact with absorbent material. The back of theslide can be wiped of excess reagents without touching the tissuesections and the slide can be air dried. To prevent tissue section fromover drying, it is highly recommended to not exceed 10 minutes for airdrying. Haematoxylin (1 ml) can be added to uniformly cover all tissuesections on the slide, and the slide can be incubated for 7 minutes atroom temperature. After Haematoxylin incubation the excess reagents canbe discarded and the back of the slide wiped as previously stated. Theslide can then be washed by immersing it gently 5 times in the 50 mlMilli-Q water, 15 times in the water in beaker 1 followed by 15 times inthe water in beaker 2. The back of the slide can be wiped of excessliquid without touching the tissue sections and the slide and placed ona flat, clean work surface. Bluing Buffer (1 ml, 10× Genomics, Inc.) canbe added to the top of the slide to uniformly cover all tissue sectionsand the slide incubated 2 minutes at room temperature. Reagents can bedrained from the slide as previously stated and the slide can be rinsed5 times by gentle immersion in the water in beaker 2. Excess liquid fromthe back of the slide can be wiped off without touching the tissuesection and the issue slide placed on a flat, clean work surface. TheEosin Mix (1 ml) can be added uniformly to the slide to cover all tissuesections and the slide can be incubated for 1 minute at roomtemperature. Excess reagent can be removed as previously stated and theslide can be immersed 15 times in the clean Milli-Q water in beaker 3.Following rinsing of the tissue slide in beaker 3, the back of thestained slide can be wiped of excess liquid and placed on a flat, cleanwork surface. The H&E stained tissue can be air dried until the tissueis opaque. Once the tissues on the slide are opaque, the tissue slidecan be once again placed on the slide adaptor on a thermocycler andincubated with the thermal cycler lid open, for 5 minutes at 37° C.Optionally, a coverslip may be mounted on the slide and the tissuesections on the slide can be imaged.

Example 14. Exemplary Methanol Fixation Workflows withImmunofluorescence Staining

In a non-limiting example, methanol fixation can be performed inconjunction with immunofluorescence staining according to the followingworkflow.

As an optional precursor step to methanol fixation forimmunofluorescence, the optimal antibody concentration can be determinedexperimentally by performing an antibody optimization protocol on tissuesections using a range of antibody concentrations. To determine theoptimal antibody concentration, eight representative frames can be drawnon a plain glass slide. One tissue slice per section can be placed ineach of the eight representative frames. Six of the frames should beused to test an antibody dilution series (e.g., antibody at 1:50, 1:100,1:200, 1:500, 1:1000, and 1:2000, or in duplicate 2 frames at 1:200, 2frames at 1:500, and 2 frames at 1:1000), and one frame can be used as a“no antibody” and one frame can be used as a “no antibody orpermeabilization enzyme” control. The antibody concentration thatresults in the specific staining of desired cells, while minimizingnonspecific background staining is selected for immunofluorescencetissue staining and additional protocol components can be furtheroptimized.

Once the tissues on a slide are methanol fixed using a method such asthat exemplified in Example 6, the slide can be placed in a cassette, orsubstrate holder, for immunofluorescence staining (FIG. 56).

A Blocking Buffer can be prepared by combining 2× Blocking Buffer (10×Genomics, Inc.) and nuclease-free water in 1:1 concentration on ice.Seventy μl of the Blocking Buffer can be added along the side of atissue containing cassette well to uniformly cover the tissue section inthat well, other wells can be similarly treated with Blocking Buffersolution. The introduction of bubbles during the addition of BlockingBuffer to the wells should be avoided. The slide can be incubated for 5minutes at room temperature. A Primary Antibody Solution is prepared bycombining 25 μl 2× Blocking Buffer with 17.3 μl nuclease-free water,along with 1 μl of primary antibody and 6.75 μl RNase Inhibitor. Fiftyμl of Primary Antibody Solution can be added along the side of each welland incubated for 30 minutes at room temperature. The Primary AntibodySolution can be removed and 100 μl of Blocking Buffer can be added alongthe side of each well. The Blocking Buffer can be used to wash thetissues and 100 μl can be added and an additional four times for a totalof five washes.

If using directly conjugated primary antibodies, the tissue is ready tobe coverslipped and imaged.

If using secondary antibodies, a Secondary Antibody Solution can beprepared by combining 25 μl 2× Blocking Buffer with 17.98 μlnuclease-free water, along with 1 μl of secondary antibody, 0.17 μlDAPI, 0.05 μl Phalloidin, and 6.75 μl RNase Inhibitor. Fifty μl of theSecondary Antibody Solution should can be added along the side of eachwell and the tissue incubated for 30 minutes at room temperature. TheSecondary Antibody Solution can be removed and 100 μl Blocking Bufferadded along the side of each well before removing the Blocking Buffer.The slide can now be prepared for mounting and coverslip application

As an exemplary method for slide mounting and coverslip application, 50ml of 3×SSC Buffer can be dispensed in a 50 ml centrifuge tube and 500ml 3×SSC Buffer can be dispensed into a beaker. The slide can be removedfrom the cassette or substrate holder and immersed in the 50 ml tubecontaining 3×SSC Buffer. One hundred and fifty μl of Mounting Medium canbe added to uniformly cover the tissue sections on the slide. Ifnecessary, the slide can be held at an angle for uniform coverage. Thecoverslip can be applied to the slide at an angle. The coverslip can beslowly lowered over the tissue, pressing down gently with forcepswithout introducing bubbles. Excess Mounting Medium can be removed byplacing one long edge of the slide on a laboratory wipe and gentlytilting the slide back and forth, repeat with the second long edge ofthe slide, and repeating the process until the coverslip is secured overthe tissue. After the coverslip is secured, imaging can proceed. Forimmunofluorescence imaging, it is not recommended that the coverslip beallowed to dry, further it is not recommended to use Cytoseal or nailpolish for securing the coverslip. The tissue may be imaged using any ofthe fluorescence imaging workflows described herein.

Example 15. Cardiac Perfusion Fixation Workflows

Tissues can be preserved and prepared for spatial analysis, wherein thetissues have been preserved by cardiac perfusion. For example, a mousebrain can be preserved by anesthetizing a mouse, perfusing with 50 mL ofchilled 1×PBS, followed by perfusing with 50 mL of chilled 4%paraformaldehyde (made with 1×PBS). The tissues of interest arecollected and placed in 4% paraformaldehyde overnight at 4° C. Thetissues are then cryoprotected by incubating the tissues in 10% sucrose(made with 1×PBS) in 4° C. for 1 hour, followed by 20% sucrose (made in1×PBS) in 4° C. for 1 hour, followed by 30% sucrose (made in 1×PBS) in4° C. overnight. After cryoprotecting the tissues, the tissues areremoved from the 30% sucrose solution and coated with OCT embeddingmedium on a petri dish before freezing the tissue. The tissue sample canthen be sectioned using a cryostat (e.g., 10 μm thick sections), and theresulting sections can be placed on a slide and warmed to promote sampleadherence to the slide. The mounted tissue samples can be stored at −80°C. until ready for use.

When ready for use, the mounted tissue samples are removed from storage,allowed to dry at 37° C. for 1 minute, and then placed in cold methanolat −20° C. for 5 minutes. After removing the tissue sample from themethanol, the tissue can be stained using any of theimmunohistochemistry or immunofluorescence workflows described herein.After performing the selected staining workflow, including imaging thestained tissue sample, the tissue sample can be further subjected toadditional workflows described herein (e.g., spatial analysisworkflows).

Example 16. Exemplary Spatial Proteomic and Genomic Analysis

An exemplary protocol for spatial proteogenomic analysis is shown inFIG. 57. To prepare a sample for spatial proteogenomic analysis, afresh-frozen tissue section mounted on a spatial analysis slide (e.g.,on an array including a plurality of capture probes, a capture probe ofthe plurality of capture probes including (i) a spatial barcode, (ii) aunique molecular identifier, and (iii) a capture domain, where thecapture domain interacts specifically with an analyte capture agent) wasdried for 1 minute at 37° C. The tissue section was fixed with methanolfor 10 minutes at −20° C. The slide was then placed in a slide holder.

The slide was rehydrated with a 1× blocking and permeabilizationsolution containing 3×SSC (saline sodium citrate), 2% (w/v) BSA (bovineserum albumin), 0.1% (v/v) Triton X-100, 1 U/μL Protector RNAseinhibitor (Roche), and 20 mM ribonucleotide vandyl complex for 5 minutesat 4° C.

The blocked slide was stained with a fluorescent primary antibody and apool of analyte capture agents (e.g., an antibody conjugated to ancapture agent barcode domain) 1:100 in 3×SSC, 2% BSA, 0.1% Triton X-100,1 U/μL for 30 minutes at room temperature. The stained slide was thenwashed five times with blocking buffer, followed by removal of the slidefrom the slide holder.

The stained slide was prepared for fluorescence imaging by mounting acoverslip using glycerol and 1 U/μL RNAse inhibitor. Fluorescenceimaging was then performed. The coverslip was removed using 3×SSC, andthe slide was placed again in the slide holder.

The tissue was treated with Proteinase K, and a spatial analysisworkflow as described herein was performed to analyze the spatiallocation of the analyte capture probes and nucleic acids released fromthe tissue.

Example 17

Understanding the cellular composition and gene expression of themammalian central nervous system (CNS) can be helpful for gaininginsights into normal, developing, and diseased neuronal tissues. Whilesingle cell RNA-seq (scRNA-seq) makes it possible to obtainhigh-resolution gene expression measurements, the technique requirescells to be dissociated from the CNS, thereby losing anatomical andorganizational information. By combining histological techniques and themassive throughput of RNA-seq, this limitation has been addressed.Unbiased capture of native mRNA was achieved using 5000 differentmolecularly barcoded, spatially encoded capture probes onto a slide overwhich tissue was placed, imaged, and permeabilized. RNAseq data was thenmapped back to image coordinates placing gene expression into contextwithin the tissue image.

Both immunofluorescent staining and oligo-conjugated antibodies(TOTALSEQ™ from BioLegend) were used to spatially resolve cell-specificproteomic markers along with gene expression in the same tissue. Thistechnique is demonstrated in this Example using serial sections of freshfrozen human cerebrum, cerebellum, and spinal cord. By aggregatingproteomic and transcriptomic data from serial sections, the resolutionof cell-type identification was improved. This “multi-omics” approachcan provide a powerful complement to traditional histopathology,enabling a greater understanding of cellular heterogeneity andorganization within the mammalian CNS. This new, more detailed view ofthe human CNS anatomy as it varies across different regions, can provideessential insight into the cell type-specific nature of neurobiology andneurodegenerative diseases.

This Example demonstrates the ability to examine anatomical andtranscriptome profiles from the same tissue section at a much higherresolution and sensitivity, at a shorter time than before (see, e.g.,Science. 2016 Jul. 1; 353(6294):78-82, incorporated herein by referencein its entirety). Also demonstrated in this Example is spatialclustering that correlates with the neuroanatomy across multiple humanCNS regions, and that the addition of immunostaining and proteindetection using analyte capture agents allows for the simultaneousexamination of protein and gene expression from the same tissue.

Spatial Gene Expression Complemented by Protein Expression

Combining immunostaining with spatial transcriptomic analysis showedgood agreement between the two techniques. In FIG. 58A-B, unbiased geneclustering of RNA transcript replicates the laminar organization of thehuman cerebral cortex (BioIVT-Asterand) following undergoing a standardspatial analysis protocol (FIG. 58B) after H&E staining (FIG. 58A) isshown. This spatial clustering (FIG. 58D) was replicated withinconsecutive sections immunostained for neurofilament H (NFH-Alexa 594,produced by hybridoma clone 801709) and glial fibrillary acidic protein(GFAP-Alexa 488, produced by hybridoma clone 644704) (FIG. 458C). InFIG. 58E-F, examination of two genes, stathmin-2 (STMN2) and RAR relatedorphan receptor B (RORB) showed specific laminar distribution betweenthe H&E stained (FIG. 58E) and immunostained samples (FIG. 58F).Combined with immunostaining, the methodology allowed for whole tissuesection analysis of both protein and the whole transcriptome. FIG. 58G-Hshow the overlap of proteomic and transcriptomic data for GFAP/GFAP(FIG. 58G) and NFH/NEFH (FIG. 58H). Scale bars=1 mm.

Use of Conjugated Antibody-Oligos for Spatial Proteogenomic Analysis

Analyte capture agents (in this case, antibodies coupled to anoligonucleotide containing an analyte capture sequence, analyte bindingmoiety barcode, and a PCR handle compatible with NGS assays)(TOTALSEQ™-A oligo-conjugated antibodies (BioLegend, San Diego)) wereused to analyze human cerebellar tissue (BioIVT-Asterand). These analytecapture agents are designed to work with any sequencing platform thatrelies on poly-dT oligonucleotides mimicking natural mRNA, thus allowingcapture by spatial analysis slides (a basic schematic is shown in FIG.59A). Immunostained samples generated robust spatial clustering,highlighting the laminar organization of the cerebellum, as shown inFIGS. 59B-C. FIG. 59B shows a merged fluorescent image of DAPI stainingof a section of human cerebellum, and FIG. 59C shows a spatialtranscriptomic analysis of the same section, overlaid on FIG. 59B). FIG.59D shows a t-SNE projection of the sequencing data illustratingcell-type clustering of the cerebellum. FIGS. 59E-I show spatial geneexpression (top) and protein staining (bottom) of astrocyte markerglutamine synthase produced by hybridoma (clone O91F4) (FIG. 59E);oligodendrocyte markers myelin CNPase (produced by hybridoma cloneSMI91) (FIG. 59F) and myelin basic protein (produced by hybridoma cloneP82H9) (FIG. 59G); stem cell marker SOX2 (produced by hybridoma clone14A6A34) (FIG. 59H); and neuronal marker SNAP-25 (produced by hybridomaclone SMI81) (FIG. 59I), each overlaid on FIG. 59B. Protein staining wascarried out using the protocol in Example 16. Scale bar=1 mm.

Multi-Omic Examination of Human Spinal Cord

Fresh frozen immunostained spinal cord samples exhibited spatialclustering (FIG. 60B) matching the neuroanatomy, especially the dorsaland ventral horns (shown as dotted lines in FIG. 60A). The proteinexpression analysis of myelin basic protein (produced by hybridoma cloneP82H9) (FIG. 60D) and SNAP-25 (produced by hybridoma clone SMI81) (FIG.60F), largely replicated the anatomy (FIG. 60A) and the correspondingspatial gene expression pattern (FIG. 60C and FIG. 60E for MBP andSNAP-25, respectively). Protein staining was carried out using theprotocol in Example 16. Scale bar=1 mm.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for preparing a biological sample for spatial proteomic analysis, the method comprising: (a) placing the biological sample on a substrate, wherein the substrate comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain; (b) contacting the biological sample with a protein blocking buffer; and (c) contacting the biological sample with a plurality of analyte capture agents, wherein an analyte capture agent of the plurality of analyte capture agents comprises: (i) an analyte binding moiety that binds to an analyte from the biological sample; (ii) an analyte binding moiety barcode; and (iii) an analyte capture sequence, wherein the analyte capture sequence hybridizes to the capture domain of the capture probe.
 2. The method of claim 1, wherein the analyte is a protein.
 3. The method of claim 1, wherein the method further comprises contacting the biological sample with methanol for about 5 minutes to about 30 minutes at about −30° C. to about 0° C., wherein said contacting is performed before step (a) or between step (a) and (b).
 4. The method of claim 1, wherein the method further comprises, after step (c): contacting the biological sample with a fluorescently-labeled antibody.
 5. The method of claim 4, wherein the method further comprises, performing fluorescence imaging of the biological sample.
 6. The method of claim 5, wherein the method further comprises, after step (c): permeabilizing the biological sample.
 7. The method of claim 7, wherein the method further comprises, contacting the biological sample with a coverslip before performing fluorescence imaging of the biological sample.
 8. The method of claim 1, wherein the analyte binding moiety is an antibody or antigen-binding fragment thereof, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, or a darpin.
 9. The method of claim 1, wherein the protein blocking buffer comprises a first RNAse inhibitor, a second RNAse inhibitor, an acid salt buffer, a non-specific blocking protein, and a surfactant.
 10. The method of claim 9, wherein the first RNAse inhibitor comprises a ribonucleotide vanadyl complex which is present in the protein blocking buffer in an amount of about 5 mM to about 50 mM.
 11. The method of claim 9, wherein the non-specific blocking protein is present in the protein blocking buffer in an amount of about 1% (w/v) to about 3% (w/v).
 12. The method of claim 9, wherein the non-specific blocking protein is a serum albumin.
 13. The method of claim 12, wherein the serum albumin is bovine serum albumin.
 14. The method of claim 9, wherein the surfactant is a non-ionic surfactant.
 15. The method of claim 14, wherein the surfactant is (C₁₄H₂₂O(C₂H₄O)_(n)).
 16. The method of claim 9, wherein the surfactant is present in the protein blocking buffer in an amount of about 0.05% to about 0.2% (w/v).
 17. The method of claim 9, wherein the second RNAse inhibitor is present in the protein blocking buffer in an amount of about 0.5 U/μL to about 5 U/μL.
 18. The method of claim 9, wherein the acid salt buffer is saline sodium citrate.
 19. The method of claim 1, wherein the biological sample is contacted with the protein blocking buffer for about 1 minute to about 10 minutes and wherein the biological sample is contacted with the protein blocking buffer at a temperature of about 1° C. to about 25° C.
 20. The method of claim 1, wherein the capture probe further comprises a unique molecular identifier, a cleavage domain, one or more functional domains, or combinations thereof.
 21. The method of claim 1, wherein the capture domain comprises a poly(T) sequence.
 22. The method of claim 1, wherein the analyte capture sequence comprises a poly(A) sequence.
 23. The method of claim 1, wherein the biological sample is a tissue sample.
 24. The method of claim 23, wherein the tissue sample is a tissue section or a fixed tissue section.
 25. The method of claim 1, wherein the substrate comprises a slide or a bead.
 26. A method of determining a location of a protein analyte in a biological sample, the method comprising: preparing a biological sample according to the method of claim 1; and sequencing (i) an analyte binding moiety barcode of the analyte capture agent or a complement thereof, and (ii) a spatial barcode of the capture probe or a complement thereof, and using the determined sequences of (i) and (ii) to correlate the location of the protein analyte in the biological sample.
 27. A method of determining a location of a protein analyte and a nucleic acid analyte in a biological sample, the method comprising: (1) preparing a biological sample according to the method of claim 1, wherein: preparing the biological sample comprises permeabilizing the biological sample, the analyte from the biological sample that is specifically bound by the analyte binding moiety of the analyte capture agent is the protein analyte, and the substrate comprises a second capture probe of the plurality of capture probes, wherein the second capture probe comprises a spatial barcode and a capture domain, wherein the capture domain of the second capture probe hybridizes to the nucleic acid analyte; (2) determining the sequence of (i) the analyte binding moiety barcode of the analyte capture agent or a complement thereof, and (ii) the spatial barcode of the capture probe or a complement thereof, and using the determined sequences of (i) and (ii) to correlate the location of the protein analyte in the biological sample; and (3) determining the sequence of (i) the nucleic acid analyte or a complement thereof, and (ii) the spatial barcode of the second capture probe or a complement thereof, and using the determined sequences of (i) and (ii) to correlate the location of the nucleic acid analyte in the biological sample.
 28. The method of claim 27, wherein the method further comprises: contacting the biological sample with a fluorescently-labeled antibody.
 29. The method of claim 27, wherein the analyte binding moiety is an antibody or antigen-binding fragment thereof, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, or a darpin.
 30. The method of claim 27, wherein the protein blocking buffer comprises a first RNAse inhibitor, a second RNAse inhibitor, an acid salt buffer, a non-specific blocking protein, and a surfactant. 